Chimeric enzymes and their applications
11760982 · 2023-09-19
Assignee
Inventors
Cpc classification
C12N2795/00022
CHEMISTRY; METALLURGY
C12Y201/01056
CHEMISTRY; METALLURGY
C12Y306/01
CHEMISTRY; METALLURGY
International classification
C12N15/73
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a chimeric enzyme comprising or consisting of at least one catalytic domain of a capping enzyme and at least one RNA-binding domain of a protein-RNA tethering system as well as its application for the production of an RNA molecule with a 5′-terminal cap.
Claims
1. A non-natural hetero-oligomeric enzyme comprising components i) through iv) linked together covalently or non-covalently: i) at least one catalytic domain of a RNA triphosphatase; ii) at least one catalytic domain of a guanylyltransferase; iii) at least one catalytic domain of a N7-guanine methyltransferase; and iv) at least one RNA-binding domain of a protein-RNA tethering system; and wherein said RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system, and wherein said non-natural hetero-oligomeric enzyme adds a cap to RNA produced by a RNA polymerase.
2. The non-natural hetero-oligomeric enzyme according to claim 1, wherein the RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein selected from the group consisting of the MS2 coat protein, the R17 coat protein and lambdoid N antitermination proteins.
3. A method for the in vitro or ex vivo production of an RNA molecule with a 5′-terminal cap, and optionally said method comprising at least one chemical modification, wherein said method comprises in vitro or ex vivo use of: A) expressing, in a host cell: an isolated nucleic acid molecule encoding a non-natural hetero-oligomeric enzyme according to claim 1; and/or B) expressing in one or more host cells a group of isolated nucleic acid molecules encoding a non-natural hetero-oligomeric enzyme according to claim 1.
4. A kit for the production of an RNA molecule with a 5′-terminal cap, comprising: i) at least one non-natural hetero-oligomeric enzyme according to claim 1; and/or ii) an isolated nucleic acid molecule encoding a non-natural hetero-oligomeric enzyme according to claim 1; and/or iii) a group of isolated nucleic acid molecules encoding a non-natural hetero-oligomeric enzyme according to claim 1.
5. A pharmaceutical composition comprising: at least one non-natural hetero-oligomeric enzyme according to claim 1.
6. A pharmaceutical composition comprising an isolated nucleic acid molecule encoding a non-natural hetero-oligomeric enzyme according to claim 1.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18) The present invention will be explained in detail with examples in the following, but the technical scope of the present invention is not limited to these examples.
Example 1: D1R/D12L, the Vaccinia Virus Capping Enzyme Tethered to Luciferase Reporter mRNA Increases its Expression
(19) 1. Objectives
(20) The objective of this experiment was to determine if the heterodimeric vaccinia virus capping enzyme appropriately tethered to Firefly Luciferase reporter mRNA synthesized in cellulo increases its expression.
(21) The vaccinia virus capping enzyme consist of two subunits, which form a heterodimer: (i) a 95 kDa subunit encoded by the vaccinia virus D1R gene (genomic sequence AY243312.1; UniProtKB/Swiss-Prot accession number P04298), designated hereafter as D1R, which has RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase enzymatic activities (Cong and Shuman 1993, Niles and Christen 1993, Higman and Niles 1994, Mao and Shuman 1994, Gong and Shuman 2003), (ii) and a 31-kDa subunit encoded by the vaccinia virus D12L gene (genomic sequence AY243312.1; UniProtKB/Swiss-Prot accession number P04318), designated hereafter as D12L, which has no intrinsic enzymatic activity, but enhances the RNA N7-guanine methyltransferase activity of the D1R subunit (Higman, Bourgeois et al. 1992, Higman, Christen et al. 1994, Mao and Shuman 1994). Cotransfection of plasmids encoding these two subunits therefore generate in cellulo the heterodimer D1R/D12L capping enzyme, which can eventually fused to a protein tethering domain.
(22) 2. Methods
(23) a. Plasmids
(24) The coding sequences of the following plasmids were optimized for expression in human cells with respect to codon adaptation index using the GeneOptimizer algorithm (Raab, Graf et al. 2010). All gene sequences were artificially synthesized and assembled from stepwise PCR using oligonucleotides, cloned and fully sequenced.
(25) For all the following examples, the conditions tested consist of a variable combination of several plasmids. In the present example, the pK1ERNAP/pT3RNAP plasmids together with Firefly luciferase reporter plasmids were used to generate in cellulo the Firefly Luciferase mRNA with or without tethering domain, which then can be specifically modified by enzyme produced by the test plasmid appropriately tethered to the Firefly Luciferase mRNA.
(26) The expression plasmids consisted of the phage T3 and K1E RNA polymerase open reading-frames (ORFs), which were subcloned in the pCMVScript plasmid backbone (Stratagene, La Jolla, Calif.), following the removal of the T7 φ10 promoter sequence. These corresponding plasmids, designated as p-followed by the name of the ORF, have the following design (i.e. pK1ERNAP or pT3RNAP;
(27) The test plasmids contained the coding sequence of the capping enzymes under investigation with (
(28) The Firefly Luciferase reporter plasmids containing the Firefly Luciferase gene under control of the K1E or T3 RNA polymerase promoters, contained a 5′-UTR sequence, Kozak consensus sequence followed by the ORF of Luciferase gene from Photinus pyralis and stop codon, RNA tethering domain consisting of four BoxBr in tandem from λ virus (optional, lacking in the untethered version; nucleotides 38312-38298 of genomic sequence of Enterobacteria phage lambda KT232076.1; SEQ ID No 7 corresponding to the nucleotide sequence of the BoxBr RNA stem-loops from A bacteriophage), poly(A) track of 40 adenosine residues, followed by a self-cleaving RNA sequence from the genomic ribozyme of the hepatitis D virus, and terminated by the bacteriophage T7 φ10 transcription stop. These plasmids were designated either pK1Ep-Luciferase/pT3p-Luciferase in their untethered versions (
(29) b. Cell Culture and Transfection
(30) For standard experiments, the Human Embryonic Kidney 293 (HEK-293, ATCC CRL 1573) were routinely grown at 37° C. in 5% CO.sub.2 atmosphere at 100% relative humidity. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4 mM L-alanyl-L-glutamine, 10% fetal bovine serum (FBS), 1% non-essential amino-acids, 1% sodium pyruvate, 1% penicillin and streptomycin, and 0.25% fungizone.
(31) Cells were routinely plated in 24-well plates at 1×10.sup.5 cells per well the day before transfection and transfected at 80% cell confluence. Transient transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) according to manufacturer's recommendations. Except otherwise stated, cells were transfected with 2 μl of Lipofectamine 2000 and 0.8 μg of total plasmid DNA. For standard luciferase and hSEAP gene reporter expression assays, cells were analyzed 48 hours after transfection, except otherwise stated.
(32) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(33) Luciferase luminescence was assayed by the Luciferase Assay System (Promega, Madison, Wis.) according to the manufacturer's recommendations. In brief, cells were lysed in Cell Culture Lysis Reagent buffer (CLR), and then centrifuged at 12,000×g for two minutes at 4° C. Luciferase Assay Reagent (Promega; 100 μl/well) diluted at 1:10 was added to supernatant (20 μl/well). Luminescence readout was taken on a Tristar 2 microplate reader (Berthold, Bad Wildbad, Germany) with a read time of one second per well.
(34) In order to normalize for transfection efficacy, cells were transfected with the pORF-eSEAP plasmid (InvivoGen, San Diego, Calif.), which encodes for the human secreted embryonic alkaline phosphatase (hSEAP) driven by the EF-1α/HTLV composite promoter. Enzymatic activity was assayed in cell culture medium using the Quanti-Blue colorimetric enzyme assay kit (InvivoGen). Gene reporter expression was expressed as the ratio of luciferase luminescence (RLU, relative light units) to eSEAP absorbance (OD, optic density).
(35) d. Statistical Analysis
(36) Statistical analyses were performed with paired two-tailed Student's t-test. Results are means (n≥4)±standard deviation. P-value<0.05 was considered statistically significant.
(37) 3. Results
(38) In this set of experiments, the Firefly Luciferase mRNA was produced by the phage K1E or T3 RNA polymerases by cotransfection of pK1ERNAP/pT3RNAP and pK1Ep-Luciferase-4xλBoxBr/pT3p-Luciferase-4xλBoxBr for the tethered version of the Firefly Luciferase reporter plasmids or pK1Ep-Luciferase-4xλBoxBr/pT3p-Luciferase-4xλBoxBr for their untethered version. The test plasmids contain the coding sequence of the D1R/D12L vaccinia virus with or without tethering domain were co-transfected. The translatability of the resulting transcripts, which is expected to increase in case of proficient capping, is measured by the Firefly Luciferase assay. A general depiction of the assay is shown
(39) Results of the first set of experiments with the K1E-driven system are shown in the table below:
(40) TABLE-US-00001 Plasmids mean SEM (1) pK1ERNAP, pK1Ep-Luciferase 35 329 3 113 (2) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBr 34 784 4 388 (3) pK1ERNAP, pD1R, pK1Ep-Luciferase- 315 159 36 188 4xλBoxBr (4) pK1ERNAP, pD12L, pK1Ep-Luciferase- 60 212 2 219 4xλBoxBr (5) pK1ERNAP, pD1R, pD12L, pK1Ep- 851 056 144 590 Luciferase-4xλBoxBr (6) pK1ERNAP, pD1R, pNλ-pD12L, pK1Ep- 2 237 689 92 709 Luciferase-4xλBoxBr (7) Baseline 14 537 3 145
(41) As expected when capping is lacking, Firefly Luciferase mRNA generated by pK1Ep-Luciferase and pK1Ep-Luciferase-4xλBoxBr cotransfected with the K1ERNAP plasmid alone (designated pK1ERNAP) was poorly expressed (row 1 and 2). Cotransfection of the untethered D1R plasmid (designated pD1R) together with pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr increased the expression by approximately 9-fold in comparison to cotransfection of pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr plasmids only (row 3 vs. 1 or 2, p<0.05, two-way Student t-test), whereas the transfection of the untethered pD12L plasmid with pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr had virtually no effect on Firefly Luciferase expression (row 4 vs. 1 or 2, p=NS, two-way Student t-test). The cotransfection of the untethered pD12L and pD1R plasmids together with pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr, which result in the vaccinia virus D1R/D12L capping enzyme heterodimer without tethering domain, significantly increased the expression of Firefly Luciferase in comparison to previous conditions, therefore confirming that mRNA capping is requested for mRNA translation (row 5 vs. 1 to 4, p=NS, two-way Student t-test p<0.05, two-way Student t-test). Finally, cotransfection of the tethered D12L plasmid (pNλ-D12L) and D1R together with pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr, which produces the tethering vaccinia virus capping enzyme D1R/Nλ-D12L, increased drastically the expression of the Luciferase mRNA by 63.3 (row 6 vs. 2) and 2.6-fold (row 6 vs. 5) in comparison to no vaccinia virus capping enzyme (pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr alone), or untethering vaccinia virus capping enzyme (pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr/pD1R/pD12L), respectively.
(42) In a second set of experiments, the Firefly Luciferase mRNA was produced by the phage T3 RNA polymerase and tethered to the vaccinia virus capping enzyme. Results of the second set of experiments with the T3-driven system are shown in the table below:
(43) TABLE-US-00002 Plasmids mean SEM (1) pT3RNAP, pT3p-Luciferase 40 387 5 041 (2) pT3RNAP, pT3p-Luciferase-4xλBoxBr 43 682 2 072 (3) pT3RNAP, pD1R, pT3p-Luciferase-4xλBoxBr 82 445 6 913 (4) pT3RNAP, pD12L, pT3p-Luciferase-4xλBoxBr 47 167 1 342 (5) pT3RNAP, pD1R, pD12L, pT3p-Luciferase- 110 371 5 390 4xλBoxBr (6) pT3RNAP, pD1R, pNA-pD12L, pT3p-Luciferase- 353 096 12 560 4xλBoxBr (7) Baseline 7 269 1 901
(44) This second set of experiments gave very similar results with cotransfection results in the following order: RNA with no 4xλBoxBr and no capping enzyme (row 1, pT3RNAP/pT3p-Luciferase) 4xλBoxBr-RNA and no capping enzyme (row 2, pT3RNAP/pT3p-Luciferase-4xλBoxBr) 4xλBoxBr-RNA with D12L subunit alone (row 4, pT3RNAP/pD12L/pT3p-Luciferase-4xλBoxBr)<4xλBoxBr-RNA with D1R (row 3, pT3RNAP/pD1R/pT3p-Luciferase-4xλBoxBr)<4xλBoxBr-RNA with untethered D1R/D12L capping enzyme (row 5, pT3RNAP/pD1R/pD12L/UpT3p-Luciferase-4xλBoxBr)<<4xλBoxBr-RNA with tethered D1R/D12L capping enzyme (row 6, pT3RNAP/pD1R/pNλ-D12L/UpT3p-Luciferase-4xλBoxBr). The expression levels of this latter condition was statistically greater than all other conditions, especially 3.2 fold higher than with the untethered D1R/D12L capping enzyme, therefore demonstrating the importance of guiding the D1R/D12L capping enzyme to the target reporter mRNA by tethering domain (p<0.05, two-way Student t-test).
(45) 4. Conclusions
(46) These experiments show that the vaccinia virus capping enzyme, which contains no known or demonstrated binding domain for a specific RNA sequence, drastically increases gene Firefly Luciferase reporter expression when appropriately tethered to uncapped and polyadenylated Firefly Luciferase reporter mRNA by the Nλ-BoxBr tethering system.
Example 2: NP868R, the African Swine Fever Virus Capping Enzyme Tethered to Luciferase Reporter mRNA Increases its Expression
(47) 1. Objectives
(48) The objective of this set of experiments was to demonstrate if NP868R, the African swine fever virus capping enzyme, appropriately tethered to polyadenylated Firefly Luciferase reporter mRNA increases its expression in cellulo. NP868R (also named G4R) is a single-unit 868 amino-acids protein, which all enzymatic activities required for cap-0 formation demonstrated in vitro, i.e. RNA-triphosphatase, RNA guanylyltransferase and RNA N7-guanine methyltransferase (Pena, Yanez et al. 1993, Jais 2011, Dixon, Chapman et al. 2013, Jais, Decroly et al. 2018).
(49) 2. Methods
(50) a. Plasmids
(51) The expression (pK1ERNAP or pT3RNAP), as well as the Firefly Luciferase reporter plasmids in their tethered versions (pK1Ep-Luciferase and pT3p-Luciferase) or untethered versions (pK1Ep-Luciferase-4xλBoxBr and pT3p-Luciferase-4xλBoxBr) were the same as described above.
(52) The test plasmid consisted of the coding sequence from the African swine fever virus NP868R capping enzyme (NCBI ASFV genomic sequence strain BA71V NC_001659; UniProtKB/Swiss-Prot accession number P32094; SEQ ID No 8 and SEQ ID No 9 corresponding to the nucleotide and amino acid sequences of African swine fever virus NP868R capping enzyme, respectively) with (pNλ-NP868R) or without the NA tethering domain (pNP868R) subcloned in the pCMVScript plasmid backbone as described above.
(53) b. Cell Culture and Transfection
(54) Same as described in Example 1.
(55) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(56) Same as described in Example 1.
(57) d. Statistical Analysis
(58) Same as described in Example 1.
(59) 3. Results
(60) The design of the assay was very similar to Example 1, except that the single subunit capping enzyme NP868R was used instead of the heterodimeric D1R/D12L capping enzyme. In brief, uncapped but polyadenylated Firefly Luciferase mRNA was synthesized in cellulo by the phage K1E or T3 RNA polymerases and its expression in presence of NP868R was assayed (
(61) Results of the first set of experiment with the K1E-driven system are shown in the table below:
(62) TABLE-US-00003 Plasmids mean SEM (1) pK1ERNAP, pK1Ep-Luciferase 109 401 7 601 (2) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBr 147 494 6 310 (3) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 2 433 576 226 803 4xλBoxBr (4) pK1ERNAP, pNλ-pNP868R, pK1Ep- 4 099 936 465 513 Luciferase-4xλBoxBr (5) Baseline 10 393 2 302
(63) In this first set of experiments, cells were cotransfected with pK1ERNAP/pK1Ep-Luciferase or pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr plasmids, showed low levels of Firefly Luciferase reporter expression (row 1 and 2). Cotransfection of the pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr with the untethered pNP868R plasmid (pNP868R) increased the expression by approximately 29-fold in comparison to pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr alone (row 3 vs. 1 or 2 respectively, p<0.05, two-way Student t-test), therefore confirming that mRNA capping is requested for mRNA translation. Finally, cotransfection of pK1ERNAP/pK1Ep-Luciferase-4xλBoxBr with tethered NP868R (pNλ-NP868R) even increased by 2.1-fold the expression of Firefly Luciferase in comparison to untethered NP868R condition, demonstrating the importance of guiding the enzyme to the target mRNA by tethering domains for proficient mRNA capping (raw 4 vs. 3, p<0.05, two-way Student t-test).
(64) Results of the second set of experiments with the T3-driven system are shown in the table below:
(65) TABLE-US-00004 Plasmids mean SEM (1) pT3RNAP, pT3p-Luciferase 75 065 7 575 (2) pT3RNAP, pT3p-Luciferase-4xλBoxBr 62 220 5 781 (3) pT3RNAP, pNP868R, pT3p-Luciferase- 122 957 24 166 4xλBoxBr (4) pT3RNAP, pNλ-NP868R, pT3p-Luciferase- 352 978 16 813 4xλBoxBr (5) Baseline 23 464 6 302
(66) In this second set of experiments, the Firefly Luciferase mRNA was produced by the phage T3 RNA polymerase and tethered to the African swine fever virus capping enzyme. This second set of experiments gave very similar results with cotransfection results in the following order: RNA with no 4xλBoxBr and no capping enzyme (row 1, pT3RNAP/pT3p-Luciferase) 4xλBoxBr-RNA without capping enzyme (row 2, pT3RNAP/pT3p-Luciferase-4xλBoxBr)<4xλBoxBr-RNA with untethered NP868R capping enzyme (row 3, pT3RNAP/pNP868R/pT3p-Luciferase-4xλBoxBr)<<4xλBoxBr-RNA with tethered NP868R capping enzyme (row 4, pT3RNAP/pNλ-NP868R/pT3p-Luciferase-4xλBoxBr). The expression levels of this last condition was statistically greater than all other conditions, especially 2.9 fold higher than with the untethered NP868R capping enzyme, therefore demonstrating the importance of tethering NP868R to the target mRNA by tethering domains for proficient mRNA capping (row 4 vs.3, p<0.05, two-way Student t-test).
(67) 4. Conclusions
(68) These experiments show that another capping enzyme, NP868R from the African swine fever virus, which contains no known or predicted binding domain for a specific RNA sequence, increases Firefly Luciferase reporter expression when appropriately tethered to uncapped and polyadenylated reporter mRNA by the Nλ-BoxBr tethering system.
Example 3: Various Protein:RNA Tethering Systems, Coupled to African Swine Fever Virus Capping Enzyme NP868R can Increase the Expression of Luciferase Reporter mRNA Produced by K1E Phage RNA Polymerase in Host-Cell Cytoplasm
(69) 1. Objectives
(70) The objectives of the present experiments were to investigate if other protein:RNA tethering systems than the Nλ-4xBoxBr system can guide the African swine fever virus capping enzyme NP868R in order to increase the expression of appropriately tethered Luciferase reporter mRNA produced by the phage K1E RNA polymerase.
(71) The following tethering systems were presently tested: i) MS2 protein and the RNA stem loop tethered sequence from MS2 virus (Valegard, Murray et al. 1994, Valegard, Murray et al. 1997), ii) NA peptide from the lambda virus and its BoxBl RNA tethered sequence (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), iii) NA peptide from the P22 lamboid virus and its BoxBr RNA tethered sequences (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), iv) NA peptide from the ϕ21 lamboid virus and its BoxBr RNA tethered sequence (Das 1993, Greenblatt, Nodwell et al. 1993, Friedman and Court 1995), v) TAT binding domain from the Human immunodeficiency virus-1 (HIV-1), which contains a biologically validated nuclear localization signal (Duconge and Toulme 1999), and TAR RNA tethered sequence (Dingwall, Ernberg et al. 1990, Weeks, Ampe et al. 1990, Karn, Dingwall et al. 1991, Puglisi, Tan et al. 1992, Frankel and Young 1998), and vi) the human small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70) protein tethering sequence (Romac, Graff et al. 1994), which contains a biologically validated nuclear localization signal (Keene, Query et al. 1999), and its U1snRNA-stem loop tethered sequence.
(72) 2. Methods
(73) a. Plasmids
(74) The pK1ERNAP expression plasmid was described in Example 1.
(75) The test plasmids consisted of the coding sequence of the African swine fever virus NP868R capping enzyme fused at its amino-terminal end to: i) bacteriophage N-antitermination protein the N-terminus of the entire MS2 protein (pMS2-NP868R, NCBI accession number NC_001417.2, UniProtKB/Swiss-Prot P03612; SEQ ID No 36 and SEQ ID No 37 corresponding to the nucleotide and amino-acid sequences, respectively), ii) N-terminal peptide from lambda bacteriophage previously described, iii)N-terminal peptide from P22 bacteriophage N-antitermination protein (pP22N-NP868R, UniProtKB/Swiss-Prot P04891), iv)N-terminal peptide from ϕ21 bacteriophage N-antitermination protein (pNϕ21-NP868R, UniProtKB/Swiss-Prot P07243), v) the TAT protein binding domain from HIV-1 isolate HXB2 (pTAT-NP868R, NCBI reference sequence: AAB50256.1), and vi) human small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70) RNA-binding protein sequence (pSNRNP70-NP868R, amino-acid 92-202, NCBI accession number NM_003089.5).
(76) The RNA tethering domains of the Firefly Luciferase reporter plasmids substituted by four tandem repeats of: i) MS2 RNA stem-loops (pK1Ep-Luciferase-4xMS2sl plasmid; nucleotides 1748-766 from Enterobacteriophage MS2 isolate DL52, NCBI accession number J0966307.1; SEQ ID No 38), ii) BoxBl RNA sequence from A virus (pK1Ep-Luciferase-4xABoxBl; NCBI accession number J02459.1 nucleotides 35518-35534), iii) λBoxBr RNA sequence from P22 lamboid virus (pK1Ep-Luciferase-4xP22BoxBr; NCBI accession number NC_002371.2, nucleotides 31,953-31,971), iv) λBoxBr RNA sequence from ϕ21 lamboid virus (pK1Ep-Luciferase-4xϕ21BoxBr; NCBI accession number AH007390.1, nucleotides 866-883), v) TAR RNA sequence from Human immunodeficiency virus type 1, isolate HXB2 (pK1Ep-Luciferase-4xTAR; NCBI accession number K03455.1, nucleotides 471-497) and vi) U1snRNA RNA stem-loop (pK1Ep-Luciferase-4xU1snRNA; NCBI accession number M28013.1, nucleotides 123-155).
(77) b. Cell Culture and Transfection
(78) Same as described in Example 1.
(79) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(80) Same as described in Example 1.
(81) d. Statistical Analysis
(82) Same as described in Example 1.
(83) 3. Results
(84) The design of the assay was very similar to Example 2, except that various protein:RNA tethering systems were tested in replacement to the Nλ:BoxBr system. In brief, uncapped Firefly Luciferase mRNA with a short polyadenylation tail of 40 adenosine residues was synthesized in cellulo by the phage K1E RNA polymerase and its expression in presence of NP868R tethered by various systems was assayed (
(85) Results of these experiments are shown in the table below:
(86) TABLE-US-00005 Plasmids mean SEM MS2-4xMS2sl tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 143 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 033 076 113 402 4xMS2sl (3) pK1ERNAP, pMS2-NP868R, pK1Ep- 1 083 076 232 757 Luciferase (4) pK1ERNAP, pMS2-NP868R, pK1Ep- 4 999 936 232 757 Luciferase-4xMS2sl (5) Baseline 162 150 Nλ-4xλBoxBl tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 433 576 113 402 4xλBoxBl (3) pK1ERNAP, pNλ-NP868R, pK1Ep- 1 430 576 232 757 Luciferase (4) pK1ERNAP, pNλ-NP868R, pK1Ep- 5 699 936 332 757 Luciferase-4xλBoxBl (5) Baseline 162 150 NP22-4xP22BoxBr tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 913 576 113 402 4xP22BoxBr (3) pK1ERNAP, pNP22-NP868R, pK1Ep- 813 576 122 757 Luciferase (4) pK1ERNAP, pNP22-NP868R, pK1Ep- 4 699 936 232 757 Luciferase-4xP22BoxBr (5) Baseline 162 150 NΦ21-4xΦ21BoxBr tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 313 576 113 402 4xΦ21BoxBr (3) pK1ERNAP, pNΦ21-NP868P, pK1Ep- 1 115 600 232 757 Luciferase (4) pK1ERNAP, pNΦ21-NP868P, pK1Ep- 4 919 936 232 757 Luciferase-4xΦ21BoxBr (5) Baseline 162 150 TAT-4xTAR tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 333 076 113 402 4xTAR (3) pK1ERNAP, pTAT-NP868R, pK1Ep- 1 153 076 232 757 Luciferase (4) pK1ERNAP, pTAT-NP868R, pK1Ep- 1 583 076 232 757 Luciferase-4xTAR (5) Baseline 162 150 SNRNP70-4xU1snRNA tethering system (1) pK1ERNAP, pNP868R, pK1Ep-Luciferase 1 233 076 113 402 (2) pK1ERNAP, pNP868R, pK1Ep-Luciferase- 1 331 222 38 402 4xU1snRNA (3) pK1ERNAP, pSNRNP70-NP868R, pK1Ep- 1 153 076 232 757 Luciferase (4) pK1ERNAP, pSNRNP70-NP868R, pK1Ep- 1 423 076 157 757 Luciferase-4xU1snRNA (5) Baseline 162 150
(87) The cotransfection of pK1ERNAP with plasmids having only one component of the tethering system, i.e. the protein domains fused to the NP868R capping enzyme of the test plasmid or Firefly Luciferase reporter plasmids with four tandem RNA tethered repeats introduced in their 3′UTR, had no significant effects on the expression of the Firefly Luciferase reporter mRNA with any system when compared to no tethering system (row 2 or 3 vs. 1; p=NS for all comparisons, two-way Student t-test). The cotransfection of pK1ERNAP with plasmids encoding for the components of the MS2-4xMS2sl (i.e. pMS2-NP868R/pK1Ep-Luciferase-4xMS2sl), Nλ-NP868R-4xABoxBl (i.e. pNλ-NP868R/pK1Ep-Luciferase-4xλBoxBl), NP22-NP868R-4xP22BoxBr (i.e. pNP22-NP868R/pK1Ep-Luciferase-4xP22BoxBr), Nϕ21-4xϕ21BoxBr (i.e. pNϕ21-NP868R/pK1Ep-Luciferase-4xϕ21BoxBr), tethering system increased significantly by 3.8- to 4.6-fold the expression levels of firefly luciferase reporter in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p<0.05 for all comparisons, two-way Student t-test). In contrast, the cotransfection of pK1ERNAP with either the TAT/4xTAR tethering system (i.e. pTAT-NP868R/pK1Ep-Luciferase-4xTAR) or the SNRNP70/4xU1snRNA tethering system (i.e. pSNRNP70-NP868R/pK1Ep-Luciferase-4xU1snRNA) shows very low change of Firefly Luciferase in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p=NS for all comparisons, two-way Student t-test).
(88) In conclusion, the best performances were obtained with the Nλ-4xλBoxBl tethering expression system (i.e. pNλ-NP868R/pK1Ep-Luciferase-4xλBoxBl), with performances of other tethering systems ranging in the following order (i.e. ratio of condition 4 vs.1): Nλ-4xλBoxBl>MS2-4xMS2sl>Nϕ21-4xϕ21BoxBr>NP22-4xP22BoxBr>>TAT-4xTAR>SNRNP70-4xU1snRNA.
(89) 4. Conclusions
(90) The present experiments show that the African swine fever virus capping enzyme NP868R can increase the expression of Firefly Luciferase mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the mRNA by bacteriophage protein-RNA tethering systems.
Example 4: Bacteriophage Protein:RNA Tethering Systems, Coupled to the D12L Subunit of the Vaccinia Virus Capping can Increase the Expression of Luciferase Reporter mRNA Produced by K1E Phage RNA Polymerase in Host-Cell Cytoplasm
(91) 1. Objectives
(92) The objectives of the present experiments were to investigate if other protein:RNA tethering systems than the Nλ-4xBoxBr system can be used to guide the heterodimeric capping enzyme from the vaccinia virus to the a target mRNA, and thereby increase its expression.
(93) The protein:RNA tethering systems tested hereinafter are the same as described in the previous example.
(94) 2. Methods
(95) a. Plasmids
(96) The pK1ERNAP expression plasmid was described in Example 1.
(97) The test plasmid consisted of the coding sequence of the D12L subunit from the vaccinia virus capping enzyme fused at its amino-terminal end with the tethering protein sequences described above. The D1R plasmid was the same as previously described.
(98) The Firefly Luciferase reporter plasmids containing the various tethered RNA sequences were the same as described in the previous example.
(99) b. Cell Culture and Transfection
(100) Same as described in Example 1.
(101) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(102) Same as described in Example 1.
(103) d. Statistical Analysis
(104) Same as described in Example 1.
(105) 3. Results
(106) The design of the assay was very similar to Example 1, except that various protein:RNA tethering systems were tested in replacement to the Nλ:BoxBr system (
(107) Results of these experiments are shown in the table below:
(108) TABLE-US-00006 Plasmids mean SEM MS2-4xMS2sl tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xMS2sl 564 522 92952 (3) pK1ERNAP, pMS2-D12L, pD1R, pK1Ep-Luciferase 687 767 84793 (4) pK1ERNAP, pMS2-D12L, pD1R, pK1Ep-Luciferase- 1 147 529 181568 4xMS2sl (5) Baseline 187 173 Nλ-4xλBoxBl tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 566 750 60984 (3) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase 521 157 161568 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase- 1 247 165 141568 4xλBoxBl (5) Baseline 187 173 NP22-4xP22BoxBr tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 748 833 41312 4xP22BoxBr (3) pK1ERNAP, pNP22-D12L, pD1R, pK1Ep-Luciferase 548 192 221568 (4) pK1ERNAP, pNP22-D12L, pD1R, pK1Ep-Luciferase- 1 306 542 127189 4xP22BoxBr (5) Baseline 187 173 NΦ21-4xΦ21BoxBr tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 576 702 123936 4xΦ21BoxBr (3) pK1ERNAP, pNΦ21-D12L, pD1R, pK1Ep-Luciferase 704 808 127189 (4) pK1ERNAP, pNΦ21-D12L, pD1R, pK1Ep-Luciferase- 1 432 734 127189 4xΦ21BoxBr (5) Baseline 187 173 TAT-4xTAR tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 573 812 41312 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase-4xTAR 489 422 81968 (3) pK1ERNAP, pTAT-D12L, pD1R, pK1Ep-Luciferase 420 064 190784 (4) pK1ERNAP, pTAT-D12L, pD1R, pK1Ep-Luciferase- 680 137 83594 4xTAR (5) Baseline 187 173 SNRNP70-4xU1snRNA tethering system (1) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase 510 718 61968 (2) pK1ERNAP, pD12L, pD1R, pK1Ep-Luciferase- 582 331 6995 4xU1snRNA (3) pK1ERNAP, pSNRNP70-D12L, pD1R, pK1Ep-Luciferase 630 096 190784 (4) pK1ERNAP, pSNRNP70-D12L, pD1R, pK1Ep-Luciferase- 618 425 129309 4xU1snRNA (5) Baseline 187 173
(109) The cotransfection of pK1ERNAP with plasmids having only one out of the two components of the tethering system, i.e. the protein domains fused to the D12L subunit of the vaccinia virus capping of the test plasmid or the Firefly Luciferase reporter plasmids with four tandem RNA tethered repeats introduced in their 3′UTR, had no significant effects on the expression of the Firefly Luciferase reporter mRNA with any system when compared to no tethering system (row 2 or 3 vs. 1; p=NS for all comparisons, two-way Student t-test). Similarly to previous findings, the cotransfection of pK1ERNAP with plasmids with all the components of the MS2-4xMS2sl and D1R subunit of the vaccinia virus capping enzyme (i.e. pMS2-D12L/pD1R/pK1Ep-Luciferase-4xMS2sl), Nλ-D12L-4xABoxBl (i.e. pNλ-D12L/pD1R/pK1Ep-Luciferase-4xABoxBl), NP22-D12L-4xP22BoxBr (i.e. pNP22-D12L/pD1R/pK1Ep-Luciferase-4xP22BoxBr), Nϕ21-4xϕ21BoxBr (i.e. pNϕ21-D12L/pD1R/pK1Ep-Luciferase-4xϕ21BoxBr), tethering system increased significantly by 2- to 2.5-fold the expression levels of firefly luciferase reporter in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (row 4 vs. 1-3; p<0.05 for all comparisons, two-way Student t-test). In contrast, the cotransfection of pK1ERNAP with either the TAT/4xTAR tethering system (i.e. pTAT-D12L/pD1R/pK1Ep-Luciferase-4xTAR) or the SNRNP70/4xU1snRNA tethering system (i.e. pSNRNP70-D12L/pD1R/pK1Ep-Luciferase-4xU1snRNA) shows very low change of Firefly Luciferase in comparison to conditions with the untethering capping enzyme and/or untethered Firefly Luciferase plasmids (p=NS for all comparisons, two-way Student t-test).
(110) Finally, the performances of the tethering systems ranged in a different order than in the previous example (i.e. ratio of condition 4 vs.1): Nϕ21-4xϕ21BoxBr>NP22-4xP22BoxBr>Nλ-4xλBoxBl>MS2-4xMS2sl>>TAT-4xTAR>SNRNP70-4xU1snRNA.
(111) 4. Conclusions
(112) The present experiments show that the heterodimeric D1R/D12L capping enzyme from the vaccinia virus can also increase the expression of Firefly Luciferase mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the mRNA by bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system.
Example 5: Fusion Between Poly(A) Polymerases and African Swine Fever Virus NP868R Capping Enzyme Increase the Expression of Luciferase Reporter mRNA Produced by K1E Phage RNA Polymerase when Appropriately Tethered to the Target Transcript
(113) 1. Objectives
(114) The present experiments aimed to determine if poly(A) polymerases fused to NP868R African Swine Fever virus capping enzyme can increase the expression of Firefly Luciferase reporter mRNA produced by the K1E phage RNA polymerase when appropriately tethered to the target transcript by the Nλ-BoxBl the thering system.
(115) 2. Methods
(116) a. Plasmids
(117) The ORFs of the following poly(A) polymerases were synthesized: i) PAP1 poly(A) polymerase from Saccharomyces cerevisiae sorted to the cytoplasm by deletion of the 42 carboxyl-terminal amino-acids that contains a nuclear localization signal (Zhelkovsky, Helmling et al. 1998) (NCBI accession number: P29468); the vaccinia virus VP55 poly(A) polymerase (UniProtKB/Swiss-Prot accession number P23371 corresponding to the nucleotide and amino-acid sequences, respectively), the viral R341 poly(A) polymerase from Acanthamoeba polyphaga mimivirus (UniProtKB/Swiss-Prot accession number: E3VZZ8), iv) the viral MG561 poly(A) polymerase from Megavirus chilensis (NCBI Accession number: YP_004894612), v) the viral C475L poly(A) polymerase from the African swine fever virus (UniProtKB/Swiss-Prot accession number: A0A0A1E081), vi) mutant PAPOLA (K656R-K657R mutation of the human PAPOLA, UniProtKB/Swiss-Prot accession number P51003) mutated at its the nuclear localization signal (Raabe, Murthy et al. 1994, Vethantham, Rao et al. 2008), vii) the wild-type canonical Mus musculus testis specific PAPOLB (UniProtKB/Swiss-Prot Q9WVP6).
(118) Four types of test plasmids were generated by in-frame subcloning of the poly(A) polymerases ORFs: i) in the pCMV-Script backbone only (e.g. pPAP1), ii) downstream to the NA tethering domain (e.g. pNλ-PAP1), iii) downstream to the Nλ-NP868R protein through a G4 flexible linker (e.g. pNλ-NP868R-G.sub.4-PAP1) resulting in the expression of monomeric protein, or iv) between the N protein tethering domain from the lambda bacteriophage and NP868R through a G4 flexible linker (e.g. pNλ-PAP1-G.sub.4-NP868R) also resulting in the expression of monomeric protein. The design of these two latter constructions is shown
(119) The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xλBoxBl) were the same as described above.
(120) b. Cell Culture and Transfection
(121) Same as described in Example 1.
(122) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(123) Same as described in Example 1.
(124) d. Statistical Analysis
(125) Same as described in Example 1.
(126) 3. Results
(127) The design of the assay was very similar to Example 2, except that the poly(A) polymerases were fused to African Swine Fever virus NP868R capping enzyme (
(128) Results of these experiments are shown in the table below:
(129) TABLE-US-00007 Plasmids mean SEM C475L poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, C475L, pK1Ep-Luciferase-4xλBoxBl 240 907 386 848 (3) pK1ERNAP, pNλ-C475L, pK1Ep-Luciferase-4xλBoxBl 301 134 499 738 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pC475L, pNλ-NP868R, pK1Ep-Luciferase- 4 246 290 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-C475L, pNλ-NP868R, pK1Ep- 6 794 064 364 911 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-C475L-G4-NP868R, pK1Ep-Luciferase- 8 685 660 414 672 4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-C475L, pK1Ep-Luciferase- 8 385 560 393 938 4xλBoxBl (9) Baseline 22 217 14 176 MG561 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, MG561, pK1Ep-Luciferase-4xλBoxBl 316 191 360 922 (3) pK1ERNAP, pNλ-MG561, pK1Ep-Luciferase-4xλBoxBl 421 588 832 896 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pMG561, pNλ-NP868R, pK1Ep-Luciferase- 4 246 290 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-MG561, pNλ-NP868R, pK1Ep- 6 199 583 364 911 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-MG561-G4-NP868R, pK1Ep-Luciferase- 8 638 575 734 672 4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-MG561, pK1Ep-Luciferase- 9 070 504 697 938 4xλBoxBl (9) Baseline 22 217 14 176 PAP1 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, PAP1, pK1Ep-Luciferase-4xλBoxBl 225 851 277 632 (3) pK1ERNAP, pNλ-PAP1, pK1Ep-Luciferase-4xλBoxBl 361 361 694 080 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAP1, pNλ-NP868R, pK1Ep-Luciferase- 4 352 447 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-PAP1, pNλ-NP868R, pK1Ep-Luciferase- 6 659 244 364 911 4xλBoxBl (7) pK1ERNAP, pNλ-PAP1-G4-NP868R, pK1Ep-Luciferase- 10 638 575 734 688 4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-PAP1, pK1Ep-Luciferase- 12 538 575 404 078 4xλBoxBl (9) Baseline 22 217 14 176 R341 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, R341, pK1Ep-Luciferase-4xλBoxBl 271 021 249 869 (3) pK1ERNAP, pNλ-R341, pK1Ep-Luciferase-4xλBoxBl 338 776 430 330 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pR341, pNλ-NP868R, pK1Ep-Luciferase- 4 069 361 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-R341, pNλ-NP868R, pK1Ep-Luciferase- 6 673 752 364 911 4xλBoxBl (7) pK1ERNAP, pNλ-R341-G4-NP868R, pK1Ep-Luciferase- 8 992 399 734 688 4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-R341, pK1Ep-Luciferase- 8 522 353 881 625 4xλBoxBl (9) Baseline 22 217 14 176 VP55 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, VP55, pK1Ep-Luciferase-4xλBoxBl 271 021 388 685 (3) pK1ERNAP, pNλ-VP55, pK1Ep-Luciferase-4xλBoxBl 331 247 291 514 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pVP55, pNλ-NP868R, pK1Ep-Luciferase- 4 493 990 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-VP55, pNλ-NP868R, pK1Ep-Luciferase- 6 673 576 364 911 4xλBoxBl (7) pK1ERNAP, pNλ-VP55-G4-NP868R, pK1Ep-Luciferase- 8 638 575 134 687 4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-VP55, pK1Ep-Luciferase- 10 107 133 220 406 4xλBoxBl (9) Baseline 22 217 14 176 PAPOLA poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 124 567 138 816 (2) pK1ERNAP, PAPOLA, pK1Ep-Luciferase-4xλBoxBl 267 819 388 685 (3) pK1ERNAP, pNλ-PAPOLA, pK1Ep-Luciferase-4xλBoxBl 274 047 291 514 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAPOLA, pNλ-NP868R, pK1Ep-Luciferase- 4 635 533 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-PAPOLA, pNλ-NP868R, pK1Ep- 7 231 432 364 911 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-PAPOLA-G4-NP868R, pK1Ep- 12 203 575 734 688 Luciferase-4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-PAPOLA, pK1Ep- 11 262 433 220 406 Luciferase-4xλBoxBl (9) Baseline 22 217 14 176 PAPOLB polu(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 150 567 138 816 (2) pK1ERNAP, PAPOLB, pK1Ep-Luciferase-4xλBoxBl 251 447 360 922 (3) pK1ERNAP, pNλ-PAPOLB, pK1Ep-Luciferase-4xλBoxBl 337 270 832 896 (4) pK1ERNAP, pNλ-NP868R, pK1Ep-Luciferase-4xλBoxBl 3 538 575 414 672 (5) pK1ERNAP, pPAPOLB, pNλ-NP868R, pK1Ep-Luciferase- 5 874 035 456 139 4xλBoxBl (6) pK1ERNAP, pNλ-PAPOLB, pNλ-NP868R, pK1Ep- 7 753 726 364 911 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-PAPOLB-G4-NP868R, pK1Ep- 9 338 575 734 688 Luciferase-4xλBoxBl (8) pK1ERNAP, pNλ-NP868R-G4-PAPOLB, pK1Ep- 9 808 575 624 484 Luciferase-4xλBoxBl (9) Baseline 22 217 14 176
(130) In the absence of mRNA capping provided by pNλ-NP868R, non-statistically significant increase of Firefly Luciferase mRNA expression of ˜1.5-fold and 2.5-fold was observed when untethered (row 2 vs. 1) or tethered poly(A) polymerase plasmids (row 3 vs. 1) were transfected, respectively (p=NS, two-way Student t-test). When the Firefly Luciferase mRNA was capped by co-transfection of pNλ-NP868R, a statistically significant increase of expression of ˜1.5-fold (row 5 vs. 4) and ˜2-fold (row 6 vs. 4) was observed when the untethered or tethered poly(A) polymerases plasmids were cotransfected, respectively (p<0.05 for all untethered poly(A) polymerases vs. no poly(A) polymerases, two-way Student t-test).
(131) Poly(A) polymerases were fused to NP868R African Swine Fever virus capping enzyme, together with the Nλ-protein tethering domain. Two types of fusion were tested with poly(A) polymerases subcloned either downstream to Nλ-NP868R through a G4 flexible linker or between the N protein tethering domain from the lambda bacteriophage and NP868R through a Ga flexible linker. All tethered fusions genes of both types increased the expression of Firefly Luciferase mRNA in comparison to non-linked enzymes (row 7 and 8 vs. 6; p<0.05 for all comparisons, two-way Student t-test). Activity of the fusion proteins ranged as follows: Nλ-NP868R-G.sub.4-C475L<Nλ-NP868R-G.sub.4-R341<Nλ-MG561-G.sub.4-NP868R<Nλ-VP55-G.sub.4-NP868R<Nλ-C475L-G.sub.4-NP868R<Nλ-R341-G.sub.4-NP868R<Nλ-NP868R-G.sub.4-MG561<Nλ-PAPOLB-G.sub.4-NP868R<Nλ-NP868R-G.sub.4-PAPOLB<Nλ-NP868R-G.sub.4-VP55<Nλ-PAP1-G.sub.4-NP868R<Nλ-NP868R-G.sub.4-PAPOLA<Nλ-PAPOLA-G.sub.4-NP868R<Nλ-NP868R-G.sub.4-PAP1
(132) 4. Conclusions
(133) The present experiments show that various poly(A) polymerases including mammalian, yeast, viral and bacterial enzymes fused to the African Swine Fever virus NP868R capping enzyme increase the expression of transcripts produced by phage RNA polymerase, when appropriately tethered with the Nλ-4xBoxBl system. Surprisingly, the fusion between various poly(A) polymerases and NP868R capping enzymes, which are not physically linked in the nature and contain no RNA-binding domain, can act synergistically and this effect is even greater when these fusion proteins are appropriately tethered (rows 7 and 8 in the above Table). These results are really surprising and one skilled in the art could have expected to obtain the same expression rate since the components are the same.
Example 6: Fusion of Poly(A) Polymerases and the D12 Subunit of the Heterodimeric Vaccinia Virus Capping Enzyme can Increase the Expression of Luciferase Reporter mRNA Produced by K1E Phage RNA Polymerase when Appropriately Tethered to the Target Transcript
(134) 1. Objectives
(135) The present experiments aim to determine if fusions of poly(A) polymerases with D12 subunit of the heterodimeric vaccinia virus capping enzyme can increase the expression of transcripts produced by phage RNA polymerase, when appropriately tethered with the Nλ-4xBoxBl system.
(136) 2. Methods
(137) a. Plasmids
(138) The pK1ERNAP expression plasmid was described in Example 1.
(139) The poly(A) polymerases tested were described in previous example and subcloned in-frame (
(140) The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xλBoxBl) were the same as described above.
(141) b. Cell Culture and Transfection
(142) Same as described in Example 1.
(143) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(144) Same as described in Example 1.
(145) d. Statistical Analysis
(146) Same as described in Example 1.
(147) 3. Results
(148) The design of the experiment was similar to previous example, except that the capping enzyme consisted of the vaccinia virus heterodimer D1R/D12 (
(149) Results of these experiments are shown in the table below:
(150) TABLE-US-00008 Plasmids mean SEM C475L poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 145 279 116 201 (2) pK1ERNAP, C475L, pK1Ep-Luciferase-4xλBoxBl 201 661 325 912 (3) pK1ERNAP, pNλ-C475L, pK1Ep-Luciferase-4xλBoxBl 253 700 466 225 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 3 301 279 429 341 (5) pK1ERNAP, pC475L, pNλ-D12L, pD1R, pK1Ep-Luciferase- 4 196 498 353 954 4xλBoxBl (6) pK1ERNAP, pNλ-C475L, pNλ-D12L, pD1R, pK1Ep- 5 272 046 95 796 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-C475L-G4-D12L, pD1R, pK1Ep-Luciferase- 7 201 395 453 620 4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-C475L, pK1Ep-Luciferase- 7 173 174 267 557 4xλBoxBl (9) Baseline 23 887 9 510 MG561 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 121 133 125 248 (2) pK1ERNAP, MG561, pK1Ep-Luciferase-4xλBoxBl 285 286 334 854 (3) pK1ERNAP, pNλ-MG561, pK1Ep-Luciferase-4xλBoxBl 391 138 866 711 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 2 240 943 218 840 (5) pK1ERNAP, pMG561, pNλ-D12L, pD1R, pK1Ep-Luciferase- 3 682 240 466 962 4xλBoxBl (6) pK1ERNAP, pNλ-MG561, pNλ-D12L, pD1R, pK1Ep- 5 346 676 374 202 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-MG561-G4-D12L, pD1R, pK1Ep- 8 858 505 603 377 Luciferase-4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-MG561, pK1Ep- 7 449 488 527 949 Luciferase-4xλBoxBl (9) Baseline 23 887 9 510 PAP1 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 120 765 132 669 (2) pK1ERNAP, PAP1, pK1Ep-Luciferase-4xλBoxBl 215 849 176 186 (3) pK1ERNAP, pNλ-PAP1, pK1Ep-Luciferase-4xλBoxBl 229 321 646 674 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 3 296 890 441 173 (5) pK1ERNAP, pPAP1, pNλ-D12L, pD1R, pK1Ep-Luciferase- 4 630 608 427 360 4xλBoxBl (6) pK1ERNAP, pNλ-PAP1, pNλ-D12L, pD1R, pK1Ep-Luciferase- 6 239 087 194 450 4xλBoxBl (7) pK1ERNAP, pNλ-PAP1-G4-D12L, pD1R, pK1Ep-Luciferase- 10 668 967 620 415 4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-PAP1, pK1Ep-Luciferase- 9 534 665 223 391 4xλBoxBl (9) Baseline 23 887 9 510 R341 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 92 989 144 738 (2) pK1ERNAP, R341, pK1Ep-Luciferase-4xλBoxBl 282 582 190 160 (3) pK1ERNAP, pNλ-R341, pK1Ep-Luciferase-4xλBoxBl 257 822 449 340 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 2 684 956 273 600 (5) pK1ERNAP, pR341, pNλ-D12L, pD1R, pK1Ep-Luciferase- 3 694 895 372 489 4xλBoxBl (6) pK1ERNAP, pNλ-R341, pNλ-D12L, pD1R, pK1Ep-Luciferase- 5 449 865 413 128 4xλBoxBl (7) pK1ERNAP, pNλ-R341-G4-D12L, pD1R, pK1Ep-Luciferase- 10 180 543 694 655 4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-R341, pK1Ep-Luciferase- 8 057 982 766 195 4xλBoxBl (9) Baseline 23 887 9 510 VP55 poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 111 045 96 804 (2) pK1ERNAP, VP55, pK1Ep-Luciferase-4xλBoxBl 188 997 328 927 (3) pK1ERNAP, pNλ-VP55, pK1Ep-Luciferase-4xλBoxBl 280 320 285 568 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 3 466 398 436 289 (5) pK1ERNAP, pVP55, pNλ-D12L, pD1R, pK1Ep-Luciferase- 4 728 261 461 933 4xλBoxBl (6) pK1ERNAP, pNλ-VP55, pNλ-D12L, pD1R, pK1Ep-Luciferase- 5 758 344 375 637 4xλBoxBl (7) pK1ERNAP, pNλ-VP55-G4-D12L, pD1R, pK1Ep-Luciferase- 7 863 085 29 635 4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-VP55, pK1Ep-Luciferase- 8 601 926 252 202 4xλBoxBl (9) Baseline 23 887 9 510 PAPOLA poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 174 916 96 128 (2) pK1ERNAP, PAPOLA, pK1Ep-Luciferase-4xλBoxBl 185 461 347 658 (3) pK1ERNAP, pNλ-PAPOLA, pK1Ep-Luciferase-4xλBoxBl 245 121 282 829 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 3 433 158 309 045 (5) pK1ERNAP, pPAPOLA, pNλ-D12L, pD1R, pK1Ep-Luciferase- 4 454 749 385 659 4xλBoxBl (6) pK1ERNAP, pNλ-PAPOLA, pNλ-D12L, pD1R, pK1Ep- 6 114 076 325 113 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-PAPOLA-G4-D12L, pD1R, pK1Ep- 9 230 982 731 066 Luciferase-4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-PAPOLA, pK1Ep- 10 284 960 221 627 Luciferase-4xλBoxBl (9) Baseline 23 887 9 510 PAPOLB poly(A) polymerase series (1) pK1ERNAP, pK1Ep-Luciferase-4xλBoxBl 134 279 130 482 (2) pK1ERNAP, PAPOLB, pK1Ep-Luciferase-4xλBoxBl 236 351 321 719 (3) pK1ERNAP, pNλ-PAPOLB, pK1Ep-Luciferase-4xλBoxBl 300 637 861 464 (4) pK1ERNAP, pNλ-D12L, pD1R, pK1Ep-Luciferase-4xλBoxBl 3 659 946 363 888 (5) pK1ERNAP, pPAPOLB, pNλ-D12L, pD1R, pK1Ep-Luciferase- 4 154 652 410 206 4xλBoxBl (6) pK1ERNAP, pNλ-PAPOLB, pNλ-D12L, pD1R, pK1Ep- 5 972 923 350 054 Luciferase-4xλBoxBl (7) pK1ERNAP, pNλ-PAPOLB-G4-D12L, pD1R, pK1Ep- 7 677 493 552 983 Luciferase-4xλBoxBl (8) pK1ERNAP, pNλ-D1R, pD12L-G4-PAPOLB, pK1Ep- 8 958 357 480 636 Luciferase-4xλBoxBl (9) Baseline 23 887 9 510
(151) In the absence of mRNA capping provided by pNλ-D12L/D1R, non-statistically significant change of Firefly Luciferase mRNA expression of ˜1.5-fold and 2-fold was observed when untethered (row 2 vs. 1) or tethered poly(A) polymerase plasmids (row 3 vs. 1) were transfected, respectively (p=NS, two-way Student t-test). Similarly to previous findings, when the Firefly Luciferase mRNA was capped by co-transfection of pNλ-D12L/D1R, a statistically significant increase of expression of ˜1.5-fold (row 5 vs. 4) and ˜2-fold (row 6 vs. 4) was observed when the untethered or tethered poly(A) polymerases plasmids were cotransfected, respectively (p<0.05 for all untethered poly(A) polymerases vs. no poly(A) polymerases, two-way Student t-test).
(152) Poly(A) polymerases were fused to the D12 subunit of the heterodimeric vaccinia virus capping enzyme, together with the Nλ-protein domain as described above. All tethered fusions genes of both types increased the expression of Firefly Luciferase mRNA in comparison to non-linked enzymes (row 7 and 8 vs. 6; p<0.05 for all comparisons, two-way Student t-test). Activity of the fusion complexes ranged as follows: Nλ-D12L/D1R-G.sub.4-C475L<Nλ-C475L-G.sub.4-D12L/D1R<Nλ-D12L/D1R-G.sub.4-MG561<Nλ-PAPOLB-G.sub.4-D12L/D1R<Nλ-VP55-G.sub.4-D12L/D1R<Nλ-D12L/D1R-G.sub.4-R341<Nλ-D12L/D1R-G.sub.4-VP55<Nλ-MG561-G.sub.4-D12L/D1R<Nλ-D12L/D1R-G.sub.4-PAPOLB<Nλ-PAPOLA-G.sub.4-D12L/D1R<Nλ-D12L/D1R-G.sub.4-PAP1<Nλ-R341-G.sub.4-D12L/D1R<Nλ-D12L/D1R-G.sub.4-PAPOLA<Nλ-PAP1-G.sub.4-D12L/D1R.
(153) 4. Conclusions
(154) The present experiments that various poly(A) polymerases fused to the D12 subunit of the heterodimeric vaccinia virus capping enzyme together with D1R subunit, which are not physically linked in the nature and contain no RNA-binding domain, can act synergistically and this effect is even greater when these fusion proteins appropriately tethered.
Example 7: Non-Covalent Tethered Coupling Between Acanthamoeba polyphaga Mimivirus R341 Poly(A) Polymerase and African Swine Fever Virus NP868R Capping Enzyme, can Increase the Expression of Luciferase Reporter mRNA Produced by Phage RNA Polymerases
(155) 1. Objectives
(156) The objectives of this set of experiments were to determine if non-covalent coupling between the R341 poly(A) polymerase and NP868R capping enzyme also results in an active expression system able to enhance the expression of uncapped Firefly Luciferase reporter mRNA when appropriately tethered. In the present experiments, the non-covalent coupling was generated using complementary leucine zippers that form heterodimers.
(157) 2. Methods
(158) a. Plasmids
(159) The pK1ERNAP expression and Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xλBoxBr) were the same as described above.
(160) Non-covalent coupling between the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme was induced by the EE.sub.1234L and RR.sub.1234L complementary leucine-zippers (SEQ ID No 28 and SEQ ID No 29 corresponding to the nucleotide and amino-acid sequences of G.sub.4-EE.sub.1234L leucine-zipper, respectively; SEQ ID No 30 and SEQ ID No 31 corresponding to the nucleotide and amino-acid sequences of RR.sub.1234L-G.sub.4 leucine-zipper, respectively). These amphipathic α-helices form an antiparallel heterodimer with dissociation affinity of ˜10.sup.−15M (Moll, Ruvinov et al. 2001). Two non-covalent heterodimeric complexes were generated between NP868R capping enzyme and R341 RNA polymerase (
(161) b. Cell Culture and Transfection
(162) Same as described in Example 1.
(163) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(164) Same as described in Example 1.
(165) d. Statistical Analysis
(166) Same as described in Example 1.
(167) 3. Results
(168) Uncapped Firefly Luciferase reporter mRNA was generated by the K1E bacteriophage RNA polymerase, using the pK1Ep-Luciferase and pK1Ep-Luciferase-4xλBoxBr plasmids as previously described (
(169) Results of these experiments are shown in the table below:
(170) TABLE-US-00009 Plasmids mean SEM (1) pK1ERNAP, pNλ-R341 497 650 182 520 (2) pK1ERNAP, pEE1234L-R341 257 650 109 512 (3) pK1ERNAP, pNλ-R341-EE1234L 522 533 146 016 (4) pK1ERNAP, pNλ-NP868R 2 377 957 237 063 (5) pK1ERNAP, pRR1234L-NP868R 1 049 099 104 587 (6) pK1ERNAP, pNλ-NP868R-RR1234L 2 331 330 232 415 (7) pK1ERNAP, pEE1234L-R341, pRR1234L- 4 539 654 325 828 NP868R (8) pK1ERNAP, pNλ-R341-EE1234L, 12 924 395 325 828 pRR1234L-NP868R (9) pK1ERNAP, pNλ-NP868R-RR1234L, 10 191 523 270 437 pEE1234L-R341 (10) Baseline 671 257
(171) The Nλ-tethering of the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase with or without leucine zipper increased modestly the expression of uncapped 4xλBoxBr-Firefly Luciferase mRNA generated by the phage K1E RNA polymerase in comparison to untethered Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase (row 1 or 3 vs. 2). The Nλ-tethering of the African Swine Fever Virus NP868R capping enzyme with or without leucine zipper increased frankly the expression of uncapped Firefly Luciferase 4xλBoxBr-mRNA generated by the phage K1E RNA polymerase in comparison to untethered NP868R capping enzyme or no capping enzyme (row 4 or 6 vs. 5).
(172) The non-covalent coupling between the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme was generated using the EE.sub.1234L and RR.sub.1234L complementary high-affinity leucine-zippers. This heterodimeric complex without the Nλ-tethering domain resulted in active complex that significantly increased the expression of uncapped Firefly Luciferase mRNA generated by the phage K1E RNA polymerase in comparison to conditions to either untethered R341 or NP868R with leucine zippers alone (row 7 vs. 2 or 5). Noticeably, the addition of Nλ-tethering at amino-terminal end of the Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase or the African swine fever virus NP868R capping enzyme of this heterodimeric complex increased by 2.80- and 2.24-fold the expression of uncapped Firefly Luciferase mRNA in comparison to the untethered complex (row 8 or 9 vs. 7; p<0.05 for both comparisons, two-way Student t-test).
(173) 4. Conclusions
(174) The present experiments show that the artificial coupling between the R341 poly(A) polymerase and the African swine fever virus NP868R capping enzyme, i.e. non-covalent through leucine zippers, also results in synergistically active heterodimers and this effect is even greater when these fusion proteins appropriately tethered.
Example 8: Assemblies Between the Acanthamoeba polyphaga mimivirus R341 Poly(A) Polymerase, African Swine Fever Virus NP868R Capping Enzyme and Phage K1E RNA Polymerase Results in Active Expression Complexes
(175) 1. Objectives
(176) The objective of the following experiment was to determine if active complexes could be generated by assembling the Acanthamoeba polyphaga mimivirus poly(A) polymerase R341, the African swine fever virus NP868R capping enzyme and the phage K1E RNA polymerase when appropriately Nλ-tethered.
(177) The assemblies tested hereinafter are designed according to the common Nλ-R341-[X1]-NP868R-[X2]-K1ERNAP protein scaffold, where [X1] and [X2] are variable.
(178) The following open-reading-frames were generated to test this hypothesis (
(179) 2. Methods
(180) a. Plasmids
(181) The Firefly Luciferase reporter plasmids in their untethered (pK1Ep-Luciferase) or tethered version (pK1Ep-Luciferase-4xλBoxBl) were the same as described above.
(182) The ORFs previously described were subcloned in the pCMVScript backbone, therefore resulting in the pNλ-R341-G.sub.4-NP868R-G.sub.4-K1ERNAP, pNλ-R341-G.sub.4-NP868R-F2A-K1ERNAP, pNλ-R341-F2A-NP868R-G.sub.4-K1ERNAP, and pNλ-R341-T2A-NP868R-G.sub.4-K1ERNAP plasmids.
(183) b. Cell Culture and Transfection
(184) Same as described in Example 1.
(185) c. Firefly Luciferase Luminescence and SEAP Colorimetric Assays
(186) Same as described in Example 1.
(187) d. Statistical Analysis
(188) Same as described in Example 1.
(189) 3. Results
(190) A depiction of the assay is shown
(191) Results of these experiments are shown in the table below:
(192) TABLE-US-00010 Plasmids mean SEM (1) pNλ-R341-G4-NP868R, pK1ERNAP, 2 989 500 321 841 pK1Ep-Luciferase-4xλBoxBl (2) pNλ-R341-G4-NP868R-G4-K1ERNAP, 3 882 614 649 466 pK1Ep-Luciferase-4xλBoxBl (3) pNλ-R341-G4-NP868R-F2A-K1ERNAP, 4 393 507 928 702 pK1Ep-Luciferase-4xλBoxBl (4) pNλ-R341-F2A-NP868R-G4-K1ERNAP, 7 778 512 219 138 pK1Ep-Luciferase-4xλBoxBl (5) pNλ-R341-T2A-NP868R-G4-K1ERNAP, 7 861 869 1 120 056 pK1Ep-Luciferase-4xλBoxBl (6) Baseline 162 088 56 504
(193) The various fusion of K1ERNAP coding sequence with Nλ-R341-F2A-NP868R were compared with non-linked Nλ-R341-F2A-NP868R and K1ERNAP. All fusions constructions gave significantly greater expression levels than non-linked Nλ-R341-F2A-NP868R and K1ERNAP (row 2-to-5 vs. 1; p<0.05, two-way Student t-test for all comparisons). Best results were obtained with Nλ-R341-T2A-NP868R-G.sub.4-K1ERNAP and Nλ-R341-F2A-NP868R-G.sub.4-K1ERNAP fusions, with other conditions ranging in the following order: Nλ-R341-T2A-NP868R-G.sub.4-K1ERNAP Nλ-R341-F2A-NP868R-G.sub.4-K1ERNAP>>Nλ-R341-G.sub.4-NP868R-F2A-K1ERNAP>Nλ-R341-G.sub.4-NP868R-G.sub.4-K1ERNAP>pNλ-R341-G.sub.4-NP868R, pK1ERNAP.
(194) 4. Conclusions
(195) The present experiments show that active tethered expression systems can be generated by assembling the poly(A) polymerase R341, African swine fever virus NP868R capping enzyme and phage K1E RNA polymerase under a Nλ-R341-[X1]-NP868R-[X2]-K1ERNAP scaffold, preferably where [X1]=T2A or F2A, and [X2]=G.sub.4. Unexpectedly, the construction Nλ-R341-F2A-NP868R-G.sub.4-K1ERNAP and Nλ-R341-T2A-NP868R-G.sub.4-K1ERNAP allow higher expression rate than the association of the constructions Nλ-R341 with NP868R-G.sub.4-K1ERNAP (row 4 and 5 vs. 1). These results are really surprising and one skilled in the art could have expected to obtain the same expression rate since the components are the same and are not physically linked in the nature and nor contain any RNA-binding domain.
BIBLIOGRAPHY
(196) Annamalai, P., S. Apte, S. Wilkens and A. L. Rao (2005). “Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints.” J Virol 79(6): 3277-3288. Ballaun, C., G. K. Farrington, M. Dobrovnik, J. Rusche, J. Hauber and E. Bohnlein (1991). “Functional analysis of human T-cell leukemia virus type I rex-response element: direct RNA binding of Rex protein correlates with in vivo activity.” J Virol 65(8): 4408-4413. Banerjee, H., A. Rahn, B. Gawande, S. Guth, J. Valcarcel and R. Singh (2004). “The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro.” RNA 10(2): 240-253. Battiste, J. L., H. Mao, N. S. Rao, R. Tan, D. R. Muhandiram, L. E. Kay, A. D. Frankel and J. R. Williamson (1996). “Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex.” Science 273(5281): 1547-1551. Bedzyk, W. D., K. M. Weidner, L. K. Denzin, L. S. Johnson, K. D. Hardman, M. W. Pantoliano, E. D. Asel and E. W. Voss, Jr. (1990). “Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.” J Biol Chem 265(30): 18615-18620. Belanger, F., J. Stepinski, E. Darzynkiewicz and J. Pelletier (2010). “Characterization of hMTr1, a human Cap1 2′-O-ribose methyltransferase.” J Biol Chem 285(43): 33037-33044. Benarroch, D., M. Jankowska-Anyszka, J. Stepinski, E. Darzynkiewicz and S. Shuman (2010). “Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities.” RNA 16(1): 211-220. Benarroch, D., Z. R. Qiu, B. Schwer and S. Shuman (2009). “Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.” RNA 15(4): 666-674. Benarroch, D., P. Smith and S. Shuman (2008). “Characterization of a trifunctional mimivirus mRNA capping enzyme and crystal structure of the RNA triphosphatase domain.” Structure 16(4): 501-512. Bird, R. E., K. D. Hardman, J. W. Jacobson, S. Johnson, B. M. Kaufman, S. M. Lee, T. Lee, S. H. Pope, G. S. Riordan and M. Whitlow (1988). “Single-chain antigen-binding proteins.” Science 242(4877): 423-426. Brisson, M., Y. He, S. Li, J. P. Yang and L. Huang (1999). “A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes.” Gene Ther 6(2): 263-270. Bujnicki, J. M. and L. Rychlewski (2001). “Reassignment of specificities of two cap methyltransferase domains in the reovirus lambda 2 protein.” Genome Biol 2(9): RESEARCH0038. Busch, R., A. Pashine, K. C. Garcia and E. D. Mellins (2002). “Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers.” J Immunol Methods 263(1-2): 111-121. Busch, R., Z. Reich, D. M. Zaller, V. Sloan and E. D. Mellins (1998). “Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM.” J Biol Chem 273(42): 27557-27564. Cai, A., M. Jankowska-Anyszka, A. Centers, L. Chlebicka, J. Stepinski, R. Stolarski, E. Darzynkiewicz and R. E. Rhoads (1999). “Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.” Biochemistry 38(26): 8538-8547. Carey, J. and O. C. Uhlenbeck (1983). “Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction.” Biochemistry 22(11): 2610-2615. Chang, H. C., Z. Bao, Y. Yao, A. G. Tse, E. C. Goyarts, M. Madsen, E. Kawasaki, P. P. Brauer, J. C. Sacchettini, S. G. Nathenson and et al. (1994). “A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments.” Proc Natl Acad Sci USA 91(24): 11408-11412. Chen, Y., H. Cai, J. Pan, N. Xiang, P. Tien, T. Ahola and D. Guo (2009). “Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase.” Proc Natl Acad Sci USA 106(9): 3484-3489. Chen, Z. and T. D. Schneider (2005). “Information theory based T7-like promoter models: classification of bacteriophages and differential evolution of promoters and their polymerases.” Nucleic Acids Res 33(19): 6172-6187. Cho, E. J., T. Takagi, C. R. Moore and S. Buratowski (1997). “mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain.” Genes Dev 11(24): 3319-3326. Choi, Y. G. and A. L. Rao (2003). “Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal.” J Virol 77(18): 9750-9757. Cilley, C. D. and J. R. Williamson (1997). “Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophoresis (PACE).” RNA 3(1): 57-67. Cong, P. and S. Shuman (1993). “Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases.” J Biol Chem 268(10): 7256-7260. Cong, P. and S. Shuman (1995). “Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.” Mol Cell Biol 15(11): 6222-6231. Cronan, J. E., Jr. (1990). “Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.” J Biol Chem 265(18): 10327-10333. Daffis, S., K. J. Szretter, J. Schriewer, J. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, Jr., P. Y. Shi and M. S. Diamond (2010). “2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members.” Nature 468(7322): 452-456. Darzynkiewicz, E., J. Stepinski, I. Ekiel, Y. Jin, D. Haber, T. Sijuwade and S. M. Tahara (1988). “Beta-globin mRNAs capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency.” Nucleic Acids Res 16(18): 8953-8962. Das, A. (1993). “Control of transcription termination by RNA-binding proteins.” Annu Rev Biochem 62: 893-930. De la Pena, M., 0. J. Kyrieleis and S. Cusack (2007). “Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase.” EMBO J 26(23): 4913-4925. Decroly, E., F. Ferron, J. Lescar and B. Canard (2011). “Conventional and unconventional mechanisms for capping viral mRNA.” Nat Rev Microbiol 10(1): 51-65. Decroly, E., I. Imbert, B. Coutard, M. Bouvet, B. Selisko, K. Alvarez, A. E. Gorbalenya, E. J. Snijder and B. Canard (2008). “Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2′O)-methyltransferase activity.” J Virol 82(16): 8071-8084. Dias, N. and C. A. Stein (2002). “Antisense oligonucleotides: basic concepts and mechanisms.” Mol Cancer Ther 1(5): 347-355. Dickson, K. S., S. R. Thompson, N. K. Gray and M. Wickens (2001). “Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.” J Biol Chem 276(45): 41810-41816. Dingwall, C., I. Ernberg, M. J. Gait, S. M. Green, S. Heaphy, J. Karn, A. D. Lowe, M. Singh and M. A. Skinner (1990). “HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.” EMBO J 9(12): 4145-4153. Dixon, L. K., D. A. Chapman, C. L. Netherton and C. Upton (2013). “African swine fever virus replication and genomics.” Virus Res 173(1): 3-14. Dong, H., D. C. Chang, M. H. Hua, S. P. Lim, Y. H. Chionh, F. Hia, Y. H. Lee, P. Kukkaro, S. M. Lok, P. C. Dedon and P. Y. Shi (2012). “2′-O methylation of internal adenosine by flavivirus NS5 methyltransferase.” PLoS Pathog 8(4): e1002642. Donnelly, M. L., G. Luke, A. Mehrotra, X. Li, L. E. Hughes, D. Gani and M. D. Ryan (2001). “Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’.” J Gen Virol 82(Pt 5): 1013-1025. Duconge, F. and J. J. Toulme (1999). “In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1.” RNA 5(12): 1605-1614. Elroy-Stein, O. and B. Moss (1990). “Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.” Proc Natl Acad Sci USA 87(17): 6743-6747. Finn, J., I. MacLachlan and P. Cullis (2005). “Factors limiting autogene-based cytoplasmic expression systems.” FASEB J 19(6): 608-610. Fortes, P., Y. Cuevas, F. Guan, P. Liu, S. Pentlicky, S. P. Jung, M. L. Martinez-Chantar, J. Prieto, D. Rowe and S. I. Gunderson (2003). “Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA.” Proc Natl Acad Sci USA 100(14): 8264-8269. Frankel, A. D. and J. A. Young (1998). “HIV-1: fifteen proteins and an RNA.” Annu Rev Biochem 67:1-25. Franklin, N. C. (1985). ““N” transcription antitermination proteins of bacteriophages lambda, phi 21 and P22.” J Mol Biol 181(1): 85-91. Friedman, D. I. and D. L. Court (1995). “Transcription antitermination: the lambda paradigm updated.” Mol Microbiol 18(2): 191-200. Furuichi, Y., A. LaFiandra and A. J. Shatkin (1977). “5′-Terminal structure and mRNA stability.” Nature 266(5599): 235-239. Furuichi, Y. and A. J. Shatkin (2000). “Viral and cellular mRNA capping: past and prospects.” Adv Virus Res 55: 135-184. Gallie, D. R. (1991). “The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.” Genes Dev 5(11): 2108-2116. Gao, X. and L. Huang (1993). “Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes.” Nucleic Acids Res 21(12): 2867-2872. Gershon, P. D., B. Y. Ahn, M. Garfield and B. Moss (1991). “Poly(A) polymerase and a dissociable polyadenylation stimulatory factor encoded by vaccinia virus.” Cell 66(6): 1269-1278. Ghosh, I., A. D. Hamilton and L. Regan (2000). “Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein.” Journal of the American Chemical Society 122(23): 5658-5659. Gingras, A. C., B. Raught and N. Sonenberg (1999). “eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.” Annu Rev Biochem 68: 913-963. Golomb, M. and M. Chamberlin (1974). “Characterization of T7-specific ribonucleic acid polymerase. IV. Resolution of the major in vitro transcripts by gel electrophoresis.” J Biol Chem 249(9): 2858-2863. Gong, C. and S. Shuman (2003). “Mapping the active site of vaccinia virus RNA triphosphatase.” Virology 309(1): 125-134. Grdzelishvili, V. Z., S. Smallwood, D. Tower, R. L. Hall, D. M. Hunt and S. A. Moyer (2005). “A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation.” J Virol 79(12): 7327-7337. Grdzelishvili, V. Z., S. Smallwood, D. Tower, R. L. Hall, D. M. Hunt and S. A. Moyer (2006). “Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation.” Virology 350(2): 394-405. Greenblatt, J., J. R. Nodwell and S. W. Mason (1993). “Transcriptional antitermination.” Nature 364(6436): 401-406. Gregoire, C., S. Y. Lin, G. Mazza, N. Rebai, I. F. Luescher and B. Malissen (1996). “Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.” Proc Natl Acad Sci USA 93(14): 7184-7189. Gu, M., K. R. Rajashankar and C. D. Lima (2010). “Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA capping apparatus.” Structure 18(2): 216-227. Gustaysson, M., J. Lehtio, S. Denman, T. T. Teeri, K. Hult and M. Martinelle (2001). “Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris.” Protein Eng 14(9): 711-715. Haline-Vaz, T., T. C. Silva and N. I. Zanchin (2008). “The human interferon-regulated ISG95 protein interacts with RNA polymerase II and shows methyltransferase activity.” Biochem Biophys Res Commun 372(4): 719-724. Han, Y. T., C. S. Tsai, Y. C. Chen, M. K. Lin, Y. H. Hsu and M. Meng (2007). “Mutational analysis of a helicase motif-based RNA 5′-triphosphatase/NTPase from bamboo mosaic virus.” Virology 367(1): 41-50. Haracska, L., R. E. Johnson, L. Prakash and S. Prakash (2005). “Trf4 and Trf5 proteins of Saccharomyces cerevisiae exhibit poly(A) RNA polymerase activity but no DNA polymerase activity.” Mol Cell Biol 25(22): 10183-10189. Hausmann, S. and S. Shuman (2005). “Giardia lamblia RNA cap guanine-N2 methyltransferase (Tgs2).” J Biol Chem 280(37): 32101-32106. Hausmann, S. and S. Shuman (2005). “Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1).” J Biol Chem 280(6): 4021-4024. Hausmann, S., S. Zheng, M. Costanzo, R. L. Brost, D. Garcin, C. Boone, S. Shuman and B. Schwer (2008). “Genetic and biochemical analysis of yeast and human cap trimethylguanosine synthase: functional overlap of 2,2,7-trimethylguanosine caps, small nuclear ribonucleoprotein components, pre-mRNA splicing factors, and RNA decay pathways.” J Biol Chem 283(46): 31706-31718. Hemmerich, P., S. Bosbach, A. von Mikecz and U. Krawinkel (1997). “Human ribosomal protein L7 binds RNA with an alpha-helical arginine-rich and lysine-rich domain.” Eur J Biochem 245(3): 549-556. Hennecke, F., C. Krebber and A. Pluckthun (1998). “Non-repetitive single-chain Fv linkers selected by selectively infective phage (SIP) technology.” Protein Eng 11(5): 405-410. Higman, M. A., N. Bourgeois and E. G. Niles (1992). “The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.” J Biol Chem 267(23): 16430-16437. Higman, M. A., L. A. Christen and E. G. Niles (1994). “The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.” J Biol Chem 269(21): 14974-14981. Higman, M. A. and E. G. Niles (1994). “Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.” J Biol Chem 269(21): 14982-14987. Hornung, V., J. Ellegast, S. Kim, K. Brzozka, A. Jung, H. Kato, H. Poeck, S. Akira, K. K. Conzelmann, M. Schlee, S. Endres and G. Hartmann (2006). “5′-Triphosphate RNA is the ligand for RIG-I.” Science 314(5801): 994-997. Hu, G., P. D. Gershon, A. E. Hodel and F. A. Quiocho (1999). “mRNA cap recognition: dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains.” Proc Natl Acad Sci USA 96(13): 7149-7154. Hu, W., F. Li, X. Yang, Z. Li, H. Xia, G. Li, Y. Wang and Z. Zhang (2004). “A flexible peptide linker enhances the immunoreactivity of two copies HBsAg preS1 (21-47) fusion protein.” J Biotechnol 107(1): 83-90. Huang, Y. and J. A. Steitz (2005). “SRprises along a messenger's journey.” Mol Cell 17(5): 613-615. Huang, Y. L., Y. T. Han, Y. T. Chang, Y. H. Hsu and M. Meng (2004). “Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus.” J Virol 78(3): 1271-1280. Huang, Y. L., Y. H. Hsu, Y. T. Han and M. Meng (2005). “mRNA guanylation catalyzed by the S-adenosylmethionine-dependent guanylyltransferase of bamboo mosaic virus.” J Biol Chem 280(13): 13153-13162. Huston, J. S., D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotny, M. N. Margolies, R. J. Ridge, R. E. Bruccoleri, E. Haber, R. Crea and et al. (1988). “Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.” Proc Natl Acad Sci USA 85(16): 5879-5883. Jais, P. (2011). Capping-prone RNA polymerase enzymes and their applications. Eukarÿs. France. Jais, P. H., E. Decroly, E. Jacquet, M. Le Boulch, A. Jais, H. Eaton, P. Ponien, F. Verdier, B. Canard, S. Gonvalves, S. Chiron, M. Le Gall, P. Mayeux and M. Shmulevitz (2018). “C3P3-G1: first generation of an artificial cytoplasmic expression system that recapitulates mRNA capping and polyadenylation.” Under review. Jiang, F., A. Gorin, W. Hu, A. Majumdar, S. Baskerville, W. Xu, A. Ellington and D. J. Patel (1999). “Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples.” Structure 7(12): 1461-1472. Kaneko, S., C. Chu, A. J. Shatkin and J. L. Manley (2007). “Human capping enzyme promotes formation of transcriptional R loops in vitro.” Proc Natl Acad Sci USA 104(45): 17620-17625. Karn, J., C. Dingwall, J. T. Finch, S. Heaphy and M. J. Gait (1991). “RNA binding by the tat and rev proteins of HIV-1.” Biochimie 73(1): 9-16. Kashiwabara, S., T. Zhuang, K. Yamagata, J. Noguchi, A. Fukamizu and T. Baba (2000). “Identification of a novel isoform of poly(A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells.” Dev Biol 228(1): 106-115. Kashiwabara, S. I., S. Tsuruta, K. Okada, Y. Yamaoka and T. Baba (2016). “Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, is essential for spermatogenesis.” J Reprod Dev 62(6): 607-614. Keene, J. D., C. C. Query and R. O. Bentley (1999). Ribonucleoproteins and RNA-binding proteins useful for the specific recognition and binding to RNA, and for control of cellular genetic processing and expression. U.S. Pat. No. 5,866,680A. Keith, J. M., M. J. Ensinger and B. Mose (1978). “HeLa cell RNA (2′-O-methyladenosine-N6-)-methyltransferase specific for the capped 5′-end of messenger RNA.” J Biol Chem 253(14): 5033-5039. Kohler, A. and E. Hurt (2007). “Exporting RNA from the nucleus to the cytoplasm.” Nat Rev Mol Cell Biol 8(10): 761-773. Komarnitsky, P., E. J. Cho and S. Buratowski (2000). “Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription.” Genes Dev 14(19): 2452-2460. Kozak, M. (2005). “Regulation of translation via mRNA structure in prokaryotes and eukaryotes.” Gene 361: 13-37. Kyriakopoulou, C. B., H. Nordvarg and A. Virtanen (2001). “A novel nuclear human poly(A) polymerase (PAP), PAP gamma.” J Biol Chem 276(36): 33504-33511. Lamla, T. and V. A. Erdmann (2004). “The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins.” Protein Expr Purif 33(1): 39-47. Langberg, S. R. and B. Moss (1981). “Post-transcriptional modifications of mRNA. Purification and characterization of cap I and cap II RNA (nucleoside-2′-)-methyltransferases from HeLa cells.” J Biol Chem 256(19): 10054-10060. LeGuyer, K. A., L. S. Behlen and O. C. Uhlenbeck (1996). “Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.” EMBO J 15(24): 6847-6853. Lee, Y. J., Y. Lee and J. H. Chung (2000). “An intronless gene encoding a poly(A) polymerase is specifically expressed in testis.” FEBS Lett 487(2): 287-292. Leppek, K. and G. Stoecklin (2014). “An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins.” Nucleic Acids Res 42(2): e13. Li, J., E. C. Fontaine-Rodriguez and S. P. Whelan (2005). “Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity.” J Virol 79(21): 13373-13384. Li, J., A. Rahmeh, M. Morelli and S. P. Whelan (2008). “A conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping.” J Virol 82(2): 775-784. Li, Y. I., Y. J. Chen, Y. H. Hsu and M. Meng (2001). “Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase.” J Virol 75(2): 782-788. Li, Y. I., T. W. Shih, Y. H. Hsu, Y. T. Han, Y. L. Huang and M. Meng (2001). “The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5′ cap structure by exhibiting RNA 5′-triphosphatase activity.” J Virol 75(24): 12114-12120. Lian, Y., M. B. De Young, A. Siwkowski, A. Hampel and J. Rappaport (1999). “The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA.” Gene Ther 6(6): 1114-1119. Lieschke, G. J., P. K. Rao, M. K. Gately and R. C. Mulligan (1997). “Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo.” Nat Biotechnol 15(1): 35-40. Lingner, J., J. Kellermann and W. Keller (1991). “Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae.” Nature 354(6353): 496-498. Liu, Z. and G. G. Carmichael (1994). “Nuclear antisense RNA. An efficient new method to inhibit gene expression.” Mol Biotechnol 2(2): 107-118. Lo, H. J., H. K. Huang and T. F. Donahue (1998). “RNA polymerase I-promoted HIS4 expression yields uncapped, polyadenylated mRNA that is unstable and inefficiently translated in Saccharomyces cerevisiae.” Mol Cell Biol 18(2): 665-675. Lugari, A., S. Betzi, E. Decroly, E. Bonnaud, A. Hermant, J. C. Guillemot, C. Debarnot, J. P. Borg, M. Bouvet, B. Canard, X. Morelli and P. Lecine (2010). “Molecular mapping of the RNA Cap 2′-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.” J Biol Chem 285(43): 33230-33241. Lumb, K. J. and P. S. Kim (1995). “A buried polar interaction imparts structural uniqueness in a designed heterodimeric coiled coil.” Biochemistry 34(27): 8642-8648. Lyakhov, D. L., B. He, X. Zhang, F. W. Studier, J. J. Dunn and W. T. McAllister (1997). “Mutant bacteriophage T7 RNA polymerases with altered termination properties.” J Mol Biol 269(1): 28-40. Makarova, 0. V., E. M. Makarov, R. Sousa and M. Dreyfus (1995). “Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed.” Proc Natl Acad Sci USA 92(26): 12250-12254. Malone, R. W., P. L. Feigner and I. M. Verma (1989). “Cationic liposome-mediated RNA transfection.” Proc Natl Acad Sci USA 86(16): 6077-6081. Mao, X., B. Schwer and S. Shuman (1995). “Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.” Mol Cell Biol 15(8): 4167-4174. Mao, X. and S. Shuman (1994). “Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer.” J Biol Chem 269(39): 24472-24479. Mao, X. and S. Shuman (1996). “Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site.” Biochemistry 35(21): 6900-6910.
(197) Martinez-Costas, J., G. Sutton, N. Ramadevi and P. Roy (1998). “Guanylyltransferase and RNA 5′-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus.” J Mol Biol 280(5): 859-866.
(198) Marzluff, W. F., E. J. Wagner and R. J. Duronio (2008). “Metabolism and regulation of canonical histone mRNAs: life without a poly(A) tail.” Nat Rev Genet 9(11): 843-854. Mauer, J., X. Luo, A. Blanjoie, X. Jiao, A. V. Grozhik, D. P. Patil, B. Linder, B. F. Pickering, J. J. Vasseur, Q. Chen, S. S. Gross, O. Elemento, F. Debart, M. Kiledjian and S. R. Jaffrey (2017). “Reversible methylation of m(6)Am in the 5′ cap controls mRNA stability.” Nature 541(7637): 371-375. McClain, D. L., H. L. Woods and M. G. Oakley (2001). “Design and characterization of a heterodimeric coiled coil that forms exclusively with an antiparallel relative helix orientation.” J Am Chem Soc 123(13): 3151-3152. McCracken, S., N. Fong, E. Rosonina, K. Yankulov, G. Brothers, D. Siderovski, A. Hessel, S. Foster, S. Shuman and D. L. Bentley (1997). “5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II.” Genes Dev 11(24): 3306-3318. Mix, H., A. V. Lobanov and V. N. Gladyshev (2007). “SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes.” Nucleic Acids Res 35(2): 414-423. Moll, J. R., S. B. Ruvinov, I. Pastan and C. Vinson (2001). “Designed heterodimerizing leucine zippers with a ranger of pls and stabilities up to 10(-15) M.” Protein Sci 10(3): 649-655. Myette, J. R. and E. G. Niles (1996). “Domain structure of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli of a subdomain possessing the RNA 5′-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme.” J Biol Chem 271(20): 11936-11944. Natalizio, B. J., N. D. Robson-Dixon and M. A. Garcia-Blanco (2009). “The Carboxyl-terminal Domain of RNA Polymerase II Is Not Sufficient to Enhance the Efficiency of Pre-mRNA Capping or Splicing in the Context of a Different Polymerase.” J Biol Chem 284(13): 8692-8702. Newton, D. L., Y. Xue, K. A. Olson, J. W. Fett and S. M. Rybak (1996). “Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains.” Biochemistry 35(2): 545-553. Niles, E. G. and L. Christen (1993). “Identification of the vaccinia virus mRNA guanyltransferase active site lysine.” J Biol Chem 268(33): 24986-24989. O'Shea, E. K., J. D. Klemm, P. S. Kim and T. Alber (1991). “X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.” Science 254(5031): 539-544. O'Shea, E. K., K. J. Lumb and P. S. Kim (1993). “Peptide ‘Velcro’: design of a heterodimeric coiled coil.” Curr Biol 3(10): 658-667. Oakley, M. G. and P. S. Kim (1998). “A buried polar interaction can direct the relative orientation of helices in a coiled coil.” Biochemistry 37(36): 12603-12610. Ogino, T. and A. K. Banerjee (2007). “Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus.” Mol Cell 25(1): 85-97. Ogino, T. and A. K. Banerjee (2008). “Formation of guanosine(5′)tetraphospho(5′)adenosine cap structure by an unconventional mRNA capping enzyme of vesicular stomatitis virus.” J Virol 82(15): 7729-7734. Ohlmann, T., M. Rau, S. J. Morley and V. M. Pain (1995). “Proteolytic cleavage of initiation factor elF-4 gamma in the reticulocyte lysate inhibits translation of capped mRNAs but enhances that of uncapped mRNAs.” Nucleic Acids Res 23(3): 334-340. Osumi-Davis, P. A., M. C. de Aguilera, R. W. Woody and A. Y. Woody (1992). “Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.” J Mol Biol 226(1): 37-45. Osumi-Davis, P. A., N. Sreerama, D. B. Volkin, C. R. Middaugh, R. W. Woody and A. Y. Woody (1994). “Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.” J Mol Biol 237(1): 5-19. Palancade, B. and O. Bensaude (2003). “Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.” Eur J Biochem 270(19): 3859-3870. Pantoliano, M. W., R. E. Bird, S. Johnson, E. D. Asel, S. W. Dodd, J. F. Wood and K. D. Hardman (1991). “Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli.” Biochemistry 30(42): 10117-10125. Pashine, A., R. Busch, M. P. Belmares, J. N. Munning, R. C. Doebele, M. Buckingham, G. P. Nolan and E. D. Mellins (2003). “Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides.” Immunity 19(2): 183-192. Pavlinkova, G., G. W. Beresford, B. J. Booth, S. K. Batra and D. Colcher (1999). “Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts.” J Nucl Med 40(9): 1536-1546. Peabody, D. S. (1993). “The RNA binding site of bacteriophage MS2 coat protein.” EMBO J 12(2): 595-600. Pena, L., R. J. Yanez, Y. Revilla, E. Vinuela and M. L. Salas (1993). “African swine fever virus guanylyltransferase.” Virology 193(1): 319-328. Pillutla, R. C., Z. Yue, E. Maldonado and A. J. Shatkin (1998). “Recombinant human mRNA cap methyltransferase binds capping enzyme/RNA polymerase IIo complexes.” J Biol Chem 273(34): 21443-21446. Puglisi, J. D., L. Chen, S. Blanchard and A. D. Frankel (1995). “Solution structure of a bovine immunodeficiency virus Tat-TAR peptide-RNA complex.” Science 270(5239): 1200-1203. Puglisi, J. D., R. Tan, B. J. Calnan, A. D. Frankel and J. R. Williamson (1992). “Conformation of the TAR RNA-arginine complex by NMR spectroscopy.” Science 257(5066): 76-80. Raab, D., M. Graf, F. Notka, T. Schodl and R. Wagner (2010). “The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization.” Syst Synth Biol 4(3): 215-225. Raabe, T., K. G. Murthy and J. L. Manley (1994). “Poly(A) polymerase contains multiple functional domains.” Mol Cell Biol 14(5): 2946-2957. Rahmeh, A. A., J. Li, P. J. Kranzusch and S. P. Whelan (2009). “Ribose 2′-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein.” J Virol 83(21): 11043-11050. Ramadevi, N., N. J. Burroughs, P. P. Mertens, I. M. Jones and P. Roy (1998). “Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus.” Proc Natl Acad Sci USA 95(23): 13537-13542. Ramadevi, N., J. Rodriguez and P. Roy (1998). “A leucine zipper-like domain is essential for dimerization and encapsidation of bluetongue virus nucleocapsid protein VP4.” J Virol 72(4): 2983-2990. Raoult, D., S. Audic, C. Robert, C. Abergel, P. Renesto, H. Ogata, B. La Scola, M. Suzan and J. M. Claverie (2004). “The 1.2-megabase genome sequence of Mimivirus.” Science 306(5700): 1344-1350. Reinisch, K. M., M. L. Nibert and S. C. Harrison (2000). “Structure of the reovirus core at 3.6 A resolution.” Nature 404(6781): 960-967. Rhoads, R. E. (1999). “Signal transduction pathways that regulate eukaryotic protein synthesis.” J Biol Chem 274(43): 30337-30340. Robert, F., M. Gagnon, D. Sans, S. Michnick and L. Brakier-Gingras (2000). “Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.” RNA 6(11): 1649-1659. Robinson, C. R. and R. T. Sauer (1998). “Optimizing the stability of single-chain proteins by linker length and composition mutagenesis.” Proc Natl Acad Sci USA 95(11): 5929-5934. Romac, J. M., D. H. Graff and J. D. Keene (1994). “The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.” Molecular and Cellular Biology 14(7): 4662-4670. Rouault, T. A. (2006). “The role of iron regulatory proteins in mammalian iron homeostasis and disease.” Nat Chem Biol 2(8): 406-414. Sacher, R. and P. Ahlquist (1989). “Effects of deletions in the N-terminal basic arm of brome mosaic virus coat protein on RNA packaging and systemic infection.” J Virol 63(11): 4545-4552. Salehi-Ashtiani, K. and J. W. Szostak (2001). “In vitro evolution suggests multiple origins for the hammerhead ribozyme.” Nature 414(6859): 82-84. Schmid, M., B. Kuchler and C. R. Eckmann (2009). “Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans.” Genes Dev 23(7): 824-836. Schmidt, T. G. and A. Skerra (1993). “The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment.” Protein Eng 6(1): 109-122. Schnierle, B. S., P. D. Gershon and B. Moss (1994). “Mutational analysis of a multifunctional protein, with mRNA 5′ cap-specific (nucleoside-2′-O-)-methyltransferase and 3′-adenylyltransferase stimulatory activities, encoded by vaccinia virus.” The Journal of biological chemistry 269(32): 20700-20706. Schroeder, S. C., B. Schwer, S. Shuman and D. Bentley (2000). “Dynamic association of capping enzymes with transcribing RNA polymerase II.” Genes Dev 14(19): 2435-2440. Schwer, B., S. Hausmann, S. Schneider and S. Shuman (2006). “Poxvirus mRNA cap methyltransferase. Bypass of the requirement for the stimulatory subunit by mutations in the catalytic subunit and evidence for intersubunit allostery.” J Biol Chem 281(28): 18953-18960. Schwer, B., N. Saha, X. Mao, H. W. Chen and S. Shuman (2000). “Structure-function analysis of yeast mRNA cap methyltransferase and high-copy suppression of conditional mutants by AdoMet synthase and the ubiquitin conjugating enzyme Cdc34p.” Genetics 155(4): 1561-1576. Shao, W. H., X. E. Zhang, H. Liu, Z. P. Zhang and A. E. Cass (2000). “Anchor-chain molecular system for orientation control in enzyme immobilization.” Bioconjug Chem 11(6): 822-826. Shi, X., P. Yao, T. Jose and P. Gershon (1996). “Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends.” RNA 2(1): 88-101. Shibagaki, Y., N. Itoh, H. Yamada, S. Nagata and K. Mizumoto (1992). “mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae.” J Biol Chem 267(14): 9521-9528. Smith, C. A., V. Calabro and A. D. Frankel (2000). “An RNA-binding chameleon.” Mol Cell 6(5): 1067-1076. Tan, R. and A. D. Frankel (1995). “Structural variety of arginine-rich RNA-binding peptides.” Proc Natl Acad Sci USA 92(12): 5282-5286. Tang, Y., N. Jiang, C. Parakh and D. Hilvert (1996). “Selection of linkers for a catalytic single-chain antibody using phage display technology.” J Biol Chem 271(26): 15682-15686. Theil, E. C. (1994). “Iron regulatory elements (IREs): a family of mRNA non-coding sequences.” Biochem J 304 (Pt 1): 1-11. Tiggemann, M., S. Jeske, M. Larsen and F. Meinhardt (2001). “Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities.” Yeast 18(9): 815-825. Ting, A. Y., K. H. Kain, R. L. Klemke and R. Y. Tsien (2001). “Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.” Proc Natl Acad Sci USA 98(26): 15003-15008. Tommasino, M., S. Ricci and C. L. Galeotti (1988). “Genome organization of the killer plasmid pGK12 from Kluyveromyces lactis.” Nucleic Acids Res 16(13): 5863-5878. Trippe, R., B. Sandrock and B. J. Benecke (1998). “A highly specific terminal uridylyl transferase modifies the 3′-end of U6 small nuclear RNA.” Nucleic Acids Res 26(13): 3119-3126. Tsukamoto, T., Y. Shibagaki, S. Imajoh-Ohmi, T. Murakoshi, M. Suzuki, A. Nakamura, H. Gotoh and K. Mizumoto (1997). “Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5′-triphosphatase, which is essential for cell viability.” Biochem Biophys Res Commun 239(1): 116-122. Tsukamoto, T., Y. Shibagaki, Y. Niikura and K. Mizumoto (1998). “Cloning and characterization of three human cDNAs encoding mRNA (guanine-7-)-methyltransferase, an mRNA cap methylase.” Biochem Biophys Res Commun 251(1): 27-34. Turner, B., S. E. Melcher, T. J. Wilson, D. G. Norman and D. M. Lilley (2005). “Induced fit of RNA on binding the L7Ae protein to the kink-turn motif.” RNA 11(8): 1192-1200. Turner, D. J., M. A. Ritter and A. J. George (1997). “Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology.” J Immunol Methods 205(1): 43-54. Valegard, K., J. B. Murray, P. G. Stockley, N. J. Stonehouse and L. Liljas (1994). “Crystal structure of an RNA bacteriophage coat protein-operator complex.” Nature 371(6498): 623-626. Valegard, K., J. B. Murray, N. J. Stonehouse, S. van den Worm, P. G. Stockley and L. Liljas (1997). “The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.” J Mol Biol 270(5): 724-738. Vethantham, V., N. Rao and J. L. Manley (2008). “Sumoylation regulates multiple aspects of mammalian poly(A) polymerase function.” Genes Dev 22(4): 499-511. Wang, L., C. R. Eckmann, L. C. Kadyk, M. Wickens and J. Kimble (2002). “A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans.” Nature 419(6904): 312-316. Weeks, K. M., C. Ampe, S. C. Schultz, T. A. Steitz and D. M. Crothers (1990). “Fragments of the HIV-1 Tat protein specifically bind TAR RNA.” Science 249(4974): 1281-1285. Wells, J. A. and D. B. Powers (1986). “In vivo formation and stability of engineered disulfide bonds in subtilisin.” J Biol Chem 261(14): 6564-6570. Whitlow, M., B. A. Bell, S. L. Feng, D. Filpula, K. D. Hardman, S. L. Hubert, M. L. Rollence, J. F. Wood, M. E. Schott, D. E. Milenic and et al. (1993). “An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability.” Protein Eng 6(8): 989-995. Wickham, T. J., M. E. Carrion and I. Kovesdi (1995). “Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs.” Gene Ther 2(10): 750-756. Wu, S. C., J. C. Yeung, P. M. Hwang and S. L. Wong (2002). “Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion.” Protein Expr Purif 24(3): 357-365. Wu, X. and L. A. Guarino (2003). “Autographa californica nucleopolyhedrovirus orf69 encodes an RNA cap (nucleoside-2′-O)-methyltransferase.” J Virol 77(6): 3430-3440. Xiao, F., W. D. Moll, S. Guo and P. Guo (2005). “Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29.” Nucleic Acids Res 33(8): 2640-2649. Yamada-Okabe, T., R. Doi, O. Shimmi, M. Arisawa and H. Yamada-Okabe (1998). “Isolation and characterization of a human cDNA for mRNA 5′-capping enzyme.” Nucleic Acids Res 26(7): 1700-1706. Ye, W., J. Yang, Q. Yu, W. Wang, J. Nancy, R. Luo and H. F. Chen (2013). “Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations.” Phys Chem Chem Phys 15(42): 18510-18522. Yi, G., R. C. Vaughan, I. Yarbrough, S. Dharmaiah and C. C. Kao (2009). “RNA binding by the brome mosaic virus capsid protein and the regulation of viral RNA accumulation.” J Mol Biol 391(2): 314-326. Yip, M. T., W. S. Dynan, P. L. Green, A. C. Black, S. J. Arrigo, A. Torbati, S. Heaphy, C. Ruland, J. D. Rosenblatt and I. S. Chen (1991). “Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.” J Virol 65(5): 2261-2272. Yu, L., A. Martins, L. Deng and S. Shuman (1997). “Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme.” J Virol 71(12): 9837-9843. Yu, L. and S. Shuman (1996). “Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.” J Virol 70(9): 6162-6168. Yue, Z., E. Maldonado, R. Pillutla, H. Cho, D. Reinberg and A. J. Shatkin (1997). “Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II.” Proc Natl Acad Sci USA 94(24): 12898-12903. Zamore, P. D., J. G. Patton and M. R. Green (1992). “Cloning and domain structure of the mammalian splicing factor U2AF.” Nature 355(6361): 609-614. Zhang, X. and F. W. Studier (1997). “Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme.” J Mol Biol 269(1): 10-27. Zhelkovsky, A., S. Helmling and C. Moore (1998). “Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires interactions at the carboxyl-terminal RNA binding domain.” Mol Cell Biol 18(10): 5942-5951. Zhu, Y., C. Qi, W. Q. Cao, A. V. Yeldandi, M. S. Rao and J. K. Reddy (2001). “Cloning and characterization of PIMT, a protein with a methyltransferase domain, which interacts with and enhances nuclear receptor coactivator PRIP function.” Proc Natl Acad Sci USA 98(18): 10380-10385.