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Andrew E Firth - One of the best experts on this subject based on the ideXlab platform.
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Programmed -2/-1 Ribosomal Frameshifting in simarteriviruses: An evolutionarily conserved mechanism
Journal of Virology, 2019Co-Authors: Yanhua Li, Sawsan Napthine, Ian Brierley, Andrew E Firth, Jens H Kuhn, Ying FangAbstract:The −2/−1 programmed Ribosomal Frameshifting (−2/−1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This −2/−1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for −2/−1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate −2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both −2 and −1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus −2/−1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in −2/−1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of −2/−1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, −2/−1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that −2/−1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication. IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved Ribosomal Frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.
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Characterization of the stimulators of protein-directed Ribosomal Frameshifting in Theiler's murine encephalomyelitis virus
Nucleic Acids Research, 2019Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, C. H. Hill, Andrew E FirthAbstract:Many viruses utilize programmed –1 Ribosomal Frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
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ASXL gain-of-function truncation mutants: defective and dysregulated forms of a natural Ribosomal Frameshifting product?
Biology Direct, 2017Co-Authors: Adam M. Dinan, John F. Atkins, Andrew E FirthAbstract:Background Programmed Ribosomal Frameshifting (PRF) is a gene expression mechanism which enables the translation of two N-terminally coincident, C-terminally distinct protein products from a single mRNA. Many viruses utilize PRF to control or regulate gene expression, but very few phylogenetically conserved examples are known in vertebrate genes. Additional sex combs-like ( ASXL ) genes 1 and 2 encode important epigenetic and transcriptional regulatory proteins that control the expression of homeotic genes during key developmental stages. Here we describe an ~150-codon overlapping ORF (termed TF ) in ASXL1 and ASXL2 that, with few exceptions, is conserved throughout vertebrates. Results Conservation of the TF ORF, strong suppression of synonymous site variation in the overlap region, and the completely conserved presence of an EH[N/S]Y motif (a known binding site for Host Cell Factor-1, HCF-1, an epigenetic regulatory factor), all indicate that TF is a protein-coding sequence. A highly conserved UCC_UUU_CGU sequence (identical to the known site of +1 Ribosomal Frameshifting for influenza virus PA-X expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL1 . Similarly, a highly conserved RG_GUC_UCU sequence (identical to a known site of −2 Ribosomal Frameshifting for arterivirus nsp2TF expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL2 . Conclusions Due to a lack of appropriate splice forms, or initiation sites, the most plausible mechanism for translation of the ASXL1 and 2 TF regions is Ribosomal Frameshifting, resulting in a transframe fusion of the N-terminal half of ASXL1 or 2 to the TF product, termed ASXL-TF. Truncation or frameshift mutants of ASXL are linked to myeloid malignancies and genetic diseases, such as Bohring-Opitz syndrome, likely at least in part as a result of gain-of-function or dominant-negative effects. Our hypothesis now indicates that these disease-associated mutant forms represent overexpressed defective versions of ASXL-TF. Reviewers This article was reviewed by Laurence Hurst and Eugene Koonin.
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protein directed Ribosomal Frameshifting temporally regulates gene expression
Nature Communications, 2017Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, Roger Ling, Leanne K Finch, Joshua D Jones, Andrew E FirthAbstract:This work was supported by Wellcome Trust grants (088789) and (106207), UK Biotechnology and Biological Research Council (BBSRC) grant (BB/J007072/1) and a European Research Council (ERC) European Union's Horizon 2020 research and innovation programme grant (646891) to A.E.F.; by BBSRC grant (BB/L000334/1) and UK Medical Research Council grant (MR/M011747/1) to I.B.; by a BBSRC PhD studentship to L.K.F.; and by a Wellcome Trust PhD scholarship to J.D.J.
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Ribosomal Frameshifting and transcriptional slippage from genetic steganography and cryptography to adventitious use
Nucleic Acids Research, 2016Co-Authors: John F. Atkins, Andrew E Firth, Gary Loughran, Pramod R Bhatt, Pavel V BaranovAbstract:Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is Frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal Frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory Frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized Frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with Ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, Frameshifting is one of the forms of recoding that enriches gene expression.
Ian Brierley - One of the best experts on this subject based on the ideXlab platform.
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Programmed -2/-1 Ribosomal Frameshifting in simarteriviruses: An evolutionarily conserved mechanism
Journal of Virology, 2019Co-Authors: Yanhua Li, Sawsan Napthine, Ian Brierley, Andrew E Firth, Jens H Kuhn, Ying FangAbstract:The −2/−1 programmed Ribosomal Frameshifting (−2/−1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This −2/−1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for −2/−1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate −2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both −2 and −1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus −2/−1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in −2/−1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of −2/−1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, −2/−1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that −2/−1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication. IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved Ribosomal Frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.
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Characterization of the stimulators of protein-directed Ribosomal Frameshifting in Theiler's murine encephalomyelitis virus
Nucleic Acids Research, 2019Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, C. H. Hill, Andrew E FirthAbstract:Many viruses utilize programmed –1 Ribosomal Frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
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protein directed Ribosomal Frameshifting temporally regulates gene expression
Nature Communications, 2017Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, Roger Ling, Leanne K Finch, Joshua D Jones, Andrew E FirthAbstract:This work was supported by Wellcome Trust grants (088789) and (106207), UK Biotechnology and Biological Research Council (BBSRC) grant (BB/J007072/1) and a European Research Council (ERC) European Union's Horizon 2020 research and innovation programme grant (646891) to A.E.F.; by BBSRC grant (BB/L000334/1) and UK Medical Research Council grant (MR/M011747/1) to I.B.; by a BBSRC PhD studentship to L.K.F.; and by a Wellcome Trust PhD scholarship to J.D.J.
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a novel role for poly c binding proteins in programmed Ribosomal Frameshifting
Nucleic Acids Research, 2016Co-Authors: Sawsan Napthine, Emmely E. Treffers, Susanne Bell, Ying Fang, Andrew E Firth, Ian Goodfellow, Eric J Snijder, Ian BrierleyAbstract:Translational control through programmed Ribosomal Frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide 'slippery' sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both -2 and -1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of Frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus-host interactions.
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Pseudoknot-Dependent Programmed —1 Ribosomal Frameshifting: Structures, Mechanisms and Models
Recoding: Expansion of Decoding Rules Enriches Gene Expression, 2009Co-Authors: Ian Brierley, Robert J.c. Gilbert, Simon PennellAbstract:Programmed —1 Ribosomal Frameshifting is a translational recoding strategy that takes place during the elongation phase of protein biosynthesis. Frameshifting occurs in response to specific signals in the mRNA; a slippery sequence, where the ribosome changes frame, and a stimulatory RNA secondary structure, usually a pseudoknot, located immediately downstream. During the frameshift the ribosome slips backwards by a single nucleotide (in the 5′-wards/—1 direction) and continues translation in the new, overlapping reading frame, generating a fusion protein composed of the products of both the original and the —1 frame coding regions. In eukaryotes, Frameshifting is largely a phenomenon of virus gene expression and associated predominantly with the expression of viral replicases. Research on Frameshifting impacts upon diverse topics, including the Ribosomal elongation cycle, RNA structure and function, tRNA modification, virus replication, antiviral intervention, evolution and bioinformatics. This chapter focuses on the structure and function of frameshift-stimulatory RNA pseudoknots and mechanistic aspects of Ribosomal Frameshifting. A variety of models of the Frameshifting process are discussed in the light of recent advances in our understanding of ribosome structure and the elongation cycle.
Sawsan Napthine - One of the best experts on this subject based on the ideXlab platform.
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Programmed -2/-1 Ribosomal Frameshifting in simarteriviruses: An evolutionarily conserved mechanism
Journal of Virology, 2019Co-Authors: Yanhua Li, Sawsan Napthine, Ian Brierley, Andrew E Firth, Jens H Kuhn, Ying FangAbstract:The −2/−1 programmed Ribosomal Frameshifting (−2/−1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This −2/−1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for −2/−1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses. However, the slippery sequences (XXXUCUCU instead of XXXUUUUU) in seven simarteriviruses can only facilitate −2 PRF to generate nsp2TF. The nsp1β of simian hemorrhagic fever virus (SHFV) was identified as a key factor that transactivates both −2 and −1 PRF, and the universally conserved Tyr111 and Arg114 in nsp1β are essential for this activity. In vitro translation experiments demonstrated the involvement of PCBPs in simarterivirus −2/−1 PRF. Using SHFV reverse genetics, we confirmed critical roles of nsp1β, slippery sequence, and C-rich motif in −2/−1 PRF in SHFV-infected cells. Attenuated virus growth ability was observed in SHFV mutants with impaired expression of nsp2TF and nsp2N. Comparative genomic sequence analysis showed that key elements of −2/−1 PRF are highly conserved in all known arteriviruses except equine arteritis virus (EAV) and wobbly possum disease virus (WPDV). Furthermore, −2/−1 PRF with SHFV PRF signal RNA can be stimulated by heterotypic nsp1βs of all non-EAV arteriviruses tested. Taken together, these data suggest that −2/−1 PRF is an evolutionarily conserved mechanism employed in non-EAV/-WPDV arteriviruses for the expression of additional viral proteins that are important for viral replication. IMPORTANCE Simarteriviruses are a group of arteriviruses infecting nonhuman primates, and a number of new species have been established in recent years. Although these arteriviruses are widely distributed among African nonhuman primates of different species, and some of them cause lethal hemorrhagic fever disease, this group of viruses has been undercharacterized. Since wild nonhuman primates are historically important sources or reservoirs of human pathogens, there is concern that simarteriviruses may be preemergent zoonotic pathogens. Thus, molecular characterization of simarteriviruses is becoming a priority in arterivirology. In this study, we demonstrated that an evolutionarily conserved Ribosomal Frameshifting mechanism is used by simarteriviruses and other distantly related arteriviruses for the expression of additional viral proteins. This mechanism is unprecedented in eukaryotic systems. Given the crucial role of ribosome function in all living systems, the potential impact of the in-depth characterization of this novel mechanism reaches beyond the field of virology.
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Characterization of the stimulators of protein-directed Ribosomal Frameshifting in Theiler's murine encephalomyelitis virus
Nucleic Acids Research, 2019Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, C. H. Hill, Andrew E FirthAbstract:Many viruses utilize programmed –1 Ribosomal Frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
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protein directed Ribosomal Frameshifting temporally regulates gene expression
Nature Communications, 2017Co-Authors: Sawsan Napthine, Susanne Bell, Ian Brierley, Roger Ling, Leanne K Finch, Joshua D Jones, Andrew E FirthAbstract:This work was supported by Wellcome Trust grants (088789) and (106207), UK Biotechnology and Biological Research Council (BBSRC) grant (BB/J007072/1) and a European Research Council (ERC) European Union's Horizon 2020 research and innovation programme grant (646891) to A.E.F.; by BBSRC grant (BB/L000334/1) and UK Medical Research Council grant (MR/M011747/1) to I.B.; by a BBSRC PhD studentship to L.K.F.; and by a Wellcome Trust PhD scholarship to J.D.J.
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a novel role for poly c binding proteins in programmed Ribosomal Frameshifting
Nucleic Acids Research, 2016Co-Authors: Sawsan Napthine, Emmely E. Treffers, Susanne Bell, Ying Fang, Andrew E Firth, Ian Goodfellow, Eric J Snijder, Ian BrierleyAbstract:Translational control through programmed Ribosomal Frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide 'slippery' sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both -2 and -1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of Frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus-host interactions.
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Transactivation of programmed Ribosomal Frameshifting by a viral protein.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Yanhua Li, Sawsan Napthine, Emmely E. Treffers, Susanne Bell, Peter A Van Veelen, Brian L. Mark, Martijn J. Van HemertAbstract:Programmed −1 Ribosomal Frameshifting (−1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes −1 Frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual −2 Frameshifting (−2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of −1 PRF, yielding a third, truncated nsp2 variant named “nsp2N.” Remarkably, we now show that both −2 and −1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β’s papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of Ribosomal Frameshifting. The newly identified Frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of Frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.
Ignacio Tinoco - One of the best experts on this subject based on the ideXlab platform.
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A Frameshifting stimulatory stem loop destabilizes the hybrid state and impedes Ribosomal translocation
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Carlos Bustamante, Ruben L Gonzalez, Ignacio TinocoAbstract:Ribosomal Frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed −1 Ribosomal Frameshifting (−1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1PRF in the Escherichia coli dnaX gene. The Frameshifting mRNA (FSmRNA) contained the Frameshifting signals: a Shine–Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of Ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large Ribosomal subunit and a Cy5-labeled tRNALys in the Ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) Ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as −1PRF.
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triplex structures in an rna pseudoknot enhance mechanical stability and increase efficiency of 1 Ribosomal Frameshifting
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Gang Chen, Carlos Bustamante, Kungyao Chang, Mingyuan Chou, Ignacio TinocoAbstract:Many viruses use programmed –1 Ribosomal Frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate Frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and Frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and Frameshifting assays to elucidate how mechanical stability of a pseudoknot and its Frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with Frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate Frameshifting. Better understanding of the molecular determinants of Frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting Frameshifting.
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a mutant rna pseudoknot that promotes Ribosomal Frameshifting in mouse mammary tumor virus
Nucleic Acids Research, 1997Co-Authors: Hunseung Kang, Ignacio TinocoAbstract:A single A-->G mutation that changes a potential A.U base pair to a G.U pair at the junction of the stems and loops of a non-Frameshifting pseudoknot dramatically increases its Frameshifting efficiency in mouse mammary tumor virus. The structure of the non-Frameshifting pseudoknot APK has been found to be very different from that of pseudoknots that cause efficient Frameshifting [Kang,H., Hines,J.V. and Tinoco,I. (1995) J. Mol. Biol. , 259, 135-147]. The 3-dimensional structure of the mutant pseudoknot was determined by restrained molecular dynamics based on NMR-derived interproton distance and torsion angle constraints. One striking feature of the mutant pseudoknot compared with the parent pseudoknot is that a G.U base pair forms at the top of stem 2, thus leaving only 1 nt at the junction of the two stems. The conformation is very different from that of the previously determined non-Frameshifting parent pseudoknot, which lacks the A.U base pair at the top of the stem and has 2 nt between the stems. However, the conformation is quite similar to that of efficient Frameshifting pseudoknots whose structures were previously determined by NMR. A single adenylate residue intervenes between the two stems and interrupts their coaxial stacking. This unpaired nucleotide produces a bent structure. The structural similarity among the efficient Frameshifting pseudoknots indicates that a specific conformation is required for Ribosomal Frameshifting, further implying a specific interaction of the pseudoknot with the ribosome.
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Structural and functional studies of retroviral RNA pseudoknots involved in Ribosomal Frameshifting: nucleotides at the junction of the two stems are important for efficient Ribosomal Frameshifting.
The EMBO journal, 1995Co-Authors: Xiaoying Chen, Ignacio Tinoco, M. Chamorro, S. I. Lee, L. X. Shen, J. V. Hines, Harold E. VarmusAbstract:Ribosomal Frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed Frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient Frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote Frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient Frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote Frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.
Lixia Yang - One of the best experts on this subject based on the ideXlab platform.
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tertiary base triple formation in the srv 1 Frameshifting pseudoknot stabilizes secondary structure components
Biochemistry, 2020Co-Authors: Lixia Yang, Desireefaye Kaixin Toh, Manchugondanahalli S Krishna, Zhensheng Zhong, Yiyao Liu, Shaomeng Wang, Yubin Gong, Gang ChenAbstract:Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA Frameshifting pseudoknot are essential in stimulating -1 Ribosomal Frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus Ribosomal Frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and Frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.
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single molecule mechanical folding and unfolding of rna hairpins effects of single a u to a c pair substitutions and single proton binding and implications for mrna structure induced 1 Ribosomal Frameshifting
Journal of the American Chemical Society, 2018Co-Authors: Lixia Yang, Zhensheng Zhong, Huan Jia, Cailing Tong, Yiran Liu, Gang ChenAbstract:A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk −1 Ribosomal Frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, −1 Frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a fra...
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Single-Molecule Mechanical Folding and Unfolding of RNA Hairpins: Effects of Single A‑U to A·C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced −1 Ribosomal Frameshifting
2018Co-Authors: Lixia Yang, Zhensheng Zhong, Huan Jia, Cailing Tong, Yiran Liu, Gang ChenAbstract:A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A+·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk −1 Ribosomal Frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, −1 Frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a Frameshifting efficiency similar to that of rHP (∼2%). Accordingly, the bottom 2–4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar Frameshifting efficiency (∼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect Frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes
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Selective Binding to mRNA Duplex Regions by Chemically Modified Peptide Nucleic Acids Stimulates Ribosomal Frameshifting
2017Co-Authors: Ru Ying Puah, Lixia Yang, Desireefaye Kaixin Toh, Huan Jia, Manikantha Maraswami, Rya Ero, Kiran M. Patil, Alan Ann Lerk Ong, Manchugondanahalli S. Krishna, Ruimin SunAbstract:Minus-one programmed Ribosomal Frameshifting (−1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one Ribosomal Frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2–8 nucleotides downstream from the slippery site. Upon −1 RF, the Ribosomal reading frame is shifted from the normal zero frame to the −1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson–Crick base pairs, respectively, through major-groove parallel PNA·RNA–RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate −1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs
