Protein-directed ribosomal frameshifting temporally regulates gene expression.
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ABSTRACT:
SUBMITTER: Napthine S
PROVIDER: S-EPMC5472766 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The expression of many genes is controlled by upstream ORFs (uORFs). Typically, the progression of the ribosome through a regulatory uORF, which depends on the physiological state of the cell, influences the expression of the downstream gene. In the classic mechanism of induction of macrolide resistance genes, antibiotics promote translation arrest within the uORF, and the static ribosome induces a conformational change in mRNA, resulting in the activation of translation of the resistance cistron. We show that ketolide antibiotics, which do not induce ribosome stalling at the uORF of the ermC resistance gene, trigger its expression via a unique mechanism. Ketolides promote frameshifting at the uORF, allowing the translating ribosome to invade the intergenic spacer. The dynamic unfolding of the mRNA structure leads to the activation of resistance. Conceptually similar mechanisms may control other cellular genes. The identified property of ketolides to reduce the fidelity of reading frame maintenance may have medical implications.
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Project description: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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Project description: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 (nsp1?). Embedded in nsp1?'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 nsp1? 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.
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Project description:Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.
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Project description:The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as 'slippery' and promotes -1 translational frameshifting. We show here, by expressing in E. coli several plant cDNAs with and without single nucleotide deletions close to the translation initiation codons, that pDEST17 is intrinsically susceptible to -1 ribosomal frameshifting at the sequence C-AAA-AAA. The deletion mutants produce correct-sized, active enzymes with a good correlation between enzyme amount and activity. We demonstrate unambiguously the frameshift through a combination of Edman degradation, MALDI-ToF mass fingerprint analysis of tryptic peptides and MALDI-ToF reflectron in-source decay (rISD) sequencing. The degree of frameshifting depends on the nature of the sequence being expressed and ranged from 25 to 60%. These findings suggest that caution should be exercised when employing pDEST17 for high-level protein expression and that the attB1 site has some potential as a tool for studying -1 frameshifting.
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Project description:MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.
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Project description:Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.
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