The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Paul J Mcdermott - One of the best experts on this subject based on the ideXlab platform.
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role of the 5 untranslated region in regulating Translational Efficiency of specific mrnas in adult cardiocytes
The FASEB Journal, 2009Co-Authors: Laura Spruill, Paul J McdermottAbstract:It has been hypothesized that Translational Efficiency is determined by the amount of secondary structure in the 5′-untranslated region (5′-UTR) of mRNA. Here, we examined whether specific 5′-UTRs ...
Daniel R Gallie - One of the best experts on this subject based on the ideXlab platform.
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coat protein enhances Translational Efficiency of alfalfa mosaic virus rnas and interacts with the eif4g component of initiation factor eif4f
Journal of General Virology, 2005Co-Authors: Ivo M Krab, Daniel R Gallie, Christian Caldwell, John F BolAbstract:The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its Translational Efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.
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Translational Efficiency is regulated by the length of the 3 untranslated region
Molecular and Cellular Biology, 1996Co-Authors: Robert L Tanguay, Daniel R GallieAbstract:All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on Translational Efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, Translational Efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the Translational Efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the Translational Efficiency and the stability of an mRNA.
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the role of the 3 untranslated region of non polyadenylated plant viral mrnas in regulating Translational Efficiency
Gene, 1994Co-Authors: Daniel R Gallie, Marilyn KobayashiAbstract:Tobacco mosaic virus (TMV) is a positive-sense RNA virus in which the single genomic RNA functions as a messenger RNA. It is a member of a class of plant viral RNAs that are the only known non-polyadenylated mRNAs in plants. The 3'-untranslated region (UTR) of TMV genomic RNA is the functional equivalent ofapoly(A) tail in that it increases mRNA stability and regulates Translational Efficiency. To determine whether the 3'-UTR of other non-polyadenylated plant viral mRNAs regulate translation, those from turnip yellow mosaic (TYMV), brome mosaic (BMV), and alfalfa mosaic (A1MV) viruses were investigated. Chimeric gene constructs were made in which the viral 3'-UTRs were introduced immediately downstream from the reporter genes encoding β-glucuronidase (GUS) and luciferase (LUC), and were translated in plant protoplasts following delivery of the mRNA using electroporation. The 3'-UTR from BMV RNA3 regulated reporter gene expression in vivo to an extent comparable to that observed for the TMV 3'-UTR. The BMV 3'-UTR increased both message stability and Translational Efficiency. As regulators of translation, the BMV and TMV 3'-UTR were dependent on the presence of a cap at the 5' terminus for function. The 3' UTR of TYMV or A1MV RNA4 had little impact on translation or transcript stability. These data suggest that although the TMV 3'-UTR is not unique in regulating translation, the 3'-UTR of plant viral mRNAs do vary in their regulatory ability.
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the cap and poly a tail function synergistically to regulate mrna Translational Efficiency
Genes & Development, 1991Co-Authors: Daniel R GallieAbstract:The cap structure and the poly(A) tail are important regulatory determinants in establishing the Translational Efficiency of a messenger RNA. Although the mechanism by which either determinant functions remains poorly characterized, the interaction between the poly(A) tail-poly(A)-binding protein complex and events occurring at the 5' terminus during translation initiation has been an intriguing possibility. In this report, the mutual dependence of the cap and the poly(A) tail was studied. Poly(A)+ and poly(A)- luciferase (Luc) mRNAs generated in vitro containing or lacking a cap were translated in vivo in tobacco protoplasts, Chinese hamster ovary cells, and yeast following delivery by electroporation. The poly(A) tail-mediated regulation of Translational Efficiency was wholly dependent on the cap for function. Moreover, cap function was enhanced over an order of magnitude by the presence of a poly(A) tail. The relative differences in stability between the mRNAs could not account for the synergism. The synergism between the cap and poly(A) tail was not observed in yeast cells in which active translation had been disrupted. In addition, the synergism was not observed in in vitro translation lysates. These data demonstrate that the cap and the poly(A) tail are interdependent for optimal function in vivo and suggest that communication between the two regulatory determinants may be important in establishing efficient translation.
Edward Darzynkiewicz - One of the best experts on this subject based on the ideXlab platform.
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phosphorothioate cap analogs increase stability and Translational Efficiency of rna vaccines in immature dendritic cells and induce superior immune responses in vivo
Gene Therapy, 2010Co-Authors: Andreas Kuhn, Joanna Kowalska, Edward Darzynkiewicz, Jacek Jemielity, Mustafa Diken, Sebastian Kreiter, Abderraouf Selmi, Christoph Huber, Ozlem Tureci, Ugur SahinAbstract:Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and Translational Efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naive antigen-specific T cells in mice. Our findings establish 5' mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.
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Phosphorothioate cap analogs stabilize mRNA and increase Translational Efficiency in mammalian cells.
RNA (New York N.Y.), 2007Co-Authors: Ewa Grudzien-nogalska, Joanna Kowalska, Edward Darzynkiewicz, Jacek Jemielity, Robert E. RhoadsAbstract:Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the α-, β-, or γ-phosphate moieties, m2 7,2′-OGpppSG, m2 7,2′-OGppSpG, and m2 7,2′-OGpSppG, respectively. The new compounds were also modified at the 2′-O position of the m7Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the α- and β-phosphates. Oligonucleotides capped with m2 7,2′-OGppSpG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m2 7,2′-OGppSpG (D1) and both isomers of m2 7,2′-OGpppSG were partially resistant. Luciferase mRNA capped with m2 7,2′-OGppSpG (D2) had a t 1/2 of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m7Gp3G-capped mRNA. Luciferase mRNAs capped with m2 7,2′-OGppSpG (D1) and m2 7,2′-OGppSpG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m7Gp3G. The greater yield of protein due to combining higher Translational Efficiency with longer t 1/2 of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.
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novel cap analogs for in vitro synthesis of mrnas with high Translational Efficiency
RNA, 2004Co-Authors: Ewa Grudzien, Edward Darzynkiewicz, Janusz Stepinski, Marzena Jankowskaanyszka, Ryszard Stolarski, Robert E. RhoadsAbstract:Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5' end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b7Gp4G, b7m(3'-O)Gp4G, and b7m2Gp4G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, Efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro Translational Efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m3(2,2,7)Gp3G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b7m2Gp4G, m7Gp3m7G, b7m(3'-OGp4G, and m7Gp4m7G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m7Gp3G, respectively. Relative Translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to Translational Efficiency.
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most mrnas in the nematode ascaris lumbricoides are trans spliced a role for spliced leader addition in Translational Efficiency
RNA, 1995Co-Authors: Patricia A Maroney, Edward Darzynkiewicz, John A Denker, Ronald Laneve, Timothy W NilsenAbstract:Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance Translational Efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.
Maria Anisimova - One of the best experts on this subject based on the ideXlab platform.
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accounting for programmed ribosomal frameshifting in the computation of codon usage bias indices
G3: Genes Genomes Genetics, 2018Co-Authors: Victor Garcia, Maria Anisimova, Stefan ZollerAbstract:Experimental evidence shows that synonymous mutations can have important consequences on genetic fitness. Many organisms display codon usage bias (CUB), where synonymous codons that are translated into the same amino acid appear with distinct frequency. Within genomes, CUB is thought to arise from selection for Translational Efficiency and accuracy, termed the Translational Efficiency hypothesis (TEH). Indeed, CUB indices correlate with protein expression levels, which is widely interpreted as evidence for Translational selection. However, these tests neglect -1 programmed ribosomal frameshifting (-1 PRF), an important Translational disruption effect found across all organisms of the tree of life. Genes that contain -1 PRF signals should cost more to express than genes without. Thus, CUB indices that do not consider -1 PRF may overestimate genes’ true adaptation to Translational Efficiency and accuracy constraints. Here, we first investigate whether -1 PRF signals do indeed carry such Translational cost. We then propose two corrections for CUB indices for genes containing -1 PRF signals. We retest the TEH in Saccharomyces cerevisiae under these corrections. We find that the correlation between corrected CUB index and protein expression remains intact for most levels of uniform -1 PRF efficiencies, and tends to increase when these efficiencies decline with protein expression. We conclude that the TEH is strengthened and that -1 PRF events constitute a promising and useful tool to examine the relationships between CUB and selection for translation Efficiency and accuracy.
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accounting for programmed ribosomal frameshifting in the computation of codon usage bias indices
bioRxiv, 2018Co-Authors: Victor Garcia, Maria AnisimovaAbstract:Experimental evidence shows that synonymous mutations can have important consequences on genetic fitness. Many organisms display codon usage bias (CUB), where synonymous codons that are translated into the same amino acid appear with distinct frequency. CUB is thought to arise from selection for Translational Efficiency and accuracy, termed the Translational Efficiency hypothesis (TEH). Indeed, CUB indices correlate with protein expression levels, which is widely interpreted as evidence for Translational selection. However, these tests neglect -1 programmed ribosomal frameshifting (-1 PRF), an important Translational disruption effect found across all organisms of the tree of life. Genes that contain -1 PRF signals should cost more to express than genes without. Thus, CUB indices that do not consider -1 PRF may overestimate genes' true adaptation to Translational Efficiency and accuracy constraints. Here, we first investigate whether -1 PRF signals do indeed carry such Translational cost. We then propose two corrections for CUB indices for genes containing -1 PRF signals. We retest the TEH under these corrections. We find that the correlation between corrected CUB index and protein expression remains intact for most levels of uniform -1 PRF efficiencies, and tends to increase when these efficiencies decline with protein expression. We conclude that the TEH is strengthened and that -1 PRF events constitute a promising and useful tool to examine the relationships between CUB and selection for translation Efficiency and accuracy.
Wenfeng Qian - One of the best experts on this subject based on the ideXlab platform.
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impact of poly a tail g content on arabidopsis pab binding and their role in enhancing Translational Efficiency
Genome Biology, 2019Co-Authors: Taolan Zhao, Qing Huan, Jing Sun, Chunyan Liu, Xiuli Hou, Ian M Silverman, Yi Zhang, Brian D Gregory, Chunming Liu, Wenfeng QianAbstract:Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and Translational Efficiency. We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding Efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding Efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding Efficiency of a gene is positively correlated with Translational Efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in Translational Efficiency when AtPAB is depleted. Our study provides a new mechanism that Translational Efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.
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Trans-splicing enhances Translational Efficiency in C. elegans.
Genome research, 2017Co-Authors: Yu-fei Yang, Xiaoqing Zhang, Taolan Zhao, Qiushi Sun, Qing Huan, Wenfeng QianAbstract:Translational Efficiency is subject to extensive regulation. However, the factors influencing such regulation are poorly understood. In Caenorhabditis elegans, 62% of genes are trans-spliced to a specific spliced leader (SL1), which replaces part of the native 5' untranslated region (5' UTR). Given the pivotal role the 5' UTR plays in the regulation of Translational Efficiency, we hypothesized that SL1 trans-splicing functions to regulate Translational Efficiency. With genome-wide analysis on Ribo-seq data, polysome profiling experiments, and CRISPR-Cas9-based genetic manipulation of trans-splicing sites, we found four lines of evidence in support of this hypothesis. First, SL1 trans-spliced genes have higher Translational efficiencies than non-trans-spliced genes. Second, SL1 trans-spliced genes have higher Translational efficiencies than non-trans-spliced orthologous genes in other nematode species. Third, an SL1 trans-spliced isoform has higher Translational Efficiency than the non-trans-spliced isoform of the same gene. Fourth, deletion of trans-splicing sites of endogenous genes leads to reduced Translational Efficiency. Importantly, we demonstrated that SL1 trans-splicing plays a key role in enhancing Translational efficiencies of essential genes. We further discovered that SL1 trans-splicing likely enhances Translational Efficiency by shortening the native 5' UTRs, hence reducing the presence of upstream start codons (uAUG) and weakening mRNA secondary structures. Taken together, our study elucidates the global function of trans-splicing in enhancing Translational Efficiency in nematodes, paving the way for further understanding the genomic mechanisms of Translational regulation.
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balanced codon usage optimizes eukaryotic Translational Efficiency
PLOS Genetics, 2012Co-Authors: Wenfeng Qian, Jianrong Yang, Nathaniel M Pearson, Calum J Maclean, Jianzhi ZhangAbstract:Cellular Efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that Translational Efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo Translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing Translational Efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on Translational Efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases Translational Efficiency, demonstrates widespread natural selection for Translational Efficiency, and offers new strategies to improve synthetic biology.
