Translon: a single term for translated regions.
Explore the source record for details and available documents.
Letter↗
Biomedical subjects
Jonathan D Dinman
Publications and source records attributed to Jonathan D Dinman.
Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae.
In viruses, programmed -1 ribosomal frameshifting (-1 PRF) signals direct the translation of alternative proteins from a single mRNA. Given that many basic regulatory mechanisms were first discovered in viral systems, the current study endeavored to: (i) identify -1 PRF signals in genomic databases, (ii) apply the protocol to the yeast genome and (iii) test selected candidates at the bench. Computational analyses revealed the presence of 10 340 consensus -1 PRF signals in the yeast genome. Of the 6353 yeast ORFs, 1275 contain at least one strong and statistically significant -1 PRF signal. Eight out of nine selected sequences promoted efficient levels of PRF in vivo. These findings provide a robust platform for high throughput computational and laboratory studies and demonstrate that functional -1 PRF signals are widespread in the genome of Saccharomyces cerevisiae. The data generated by this study have been deposited into a publicly available database called the PRFdb. The presence of stable mRNA pseudoknot structures in these -1 PRF signals, and the observation that the predicted outcomes of nearly all of these genomic frameshift signals would direct ribosomes to premature termination codons, suggest two possible mRNA destabilization pathways through which -1 PRF signals could post-transcriptionally regulate mRNA abundance.
Base Sequence↗
An arc of unpaired "hinge bases" facilitates information exchange among functional centers of the ribosome.
Information must be shared and functions coordinated among the spatially distinct functional centers of the ribosome. To address these issues, a yeast-based genetic system enabling generation of stable strains expressing only mutant forms of rRNA was devised. The B1a bridge (helix 38) has been implicated in the subtle modulation of numerous ribosomal functions. Base-specific mutations were introduced into helix 38 at sites affecting the B1a bridge and where it contacts the aminoacyl-tRNA (aa-tRNA) D-loop. Both sets of mutants promoted increased affinities for aa-tRNA but had different effects in their responses to two A-site-specific drugs and on suppression nonsense codons. Structural analyses revealed an arc of nucleotides in 25S rRNA that link the B1a bridge, the peptidyltransferase center, the GTPase-associated center, and the sarcin/ricin loop. We propose that a series of regularly spaced "hinge bases" provide fulcrums around which rigid helices can reorient themselves depending on the occupancy status of the A-site.
Base Sequence↗
Efficient expression of the 15-kDa form of infectious pancreatic necrosis virus VP5 by suppression of a UGA codon.
Infectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae family, encodes a nonstructural VP5 protein from a small open reading frame (ORF), which overlaps with a major ORF encoding pVP2, VP4 and VP3 proteins. In majority of the Sp strains of IPNV sequenced to date, VP5 gene codes for a 15-kDa protein. However, we have shown that in highly virulent strains, there is a premature in-frame stop codon (UGA) at nucleotide (nt) position 427, (preceding the 15-kDa stop codon at nt position 511) which could encode a 12-kDa protein. Using reverse genetics, we recovered recombinant rNVI15, rNVI15-15K and rNVI15-DeltaVP5 viruses (which could encode 12 or 15-kDa VP5 or lack the expression of VP5, respectively) and demonstrated that VP5 is dispensable for viral replication in vivo but is not involved in virulence (Santi, N., Song, H., Vakharia, V. N., Evensen, Ø., 2005a. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79, 9206-9216). Here, we utilized these viruses to investigate the gene expression of VP5 in vitro. Our results indicate that a 15-kDa VP5 is produced in rNVI15-infected cells, albeit at lower levels than in rNVI15-15K-infected cells, suggesting that the opal stop codon at nt 427 is suppressed. Furthermore, to examine translational suppression of the opal stop codon in VP5 gene, we constructed plasmids containing VP5-specific sequence and employed a yeast-based bicistronic dual-luciferase reporter system (Harger, J.W., Dinman, J.D., 2003. An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. RNA 9, 1019-1024). Our results demonstrate that the VP5 sequence (with or without a stop codon) yielded approximately 13% termination suppression and the efficiency is directly related to the base immediately 3' of the termination codon, C>A>U>G.
Animals↗
Comparative study of the effects of heptameric slippery site composition on -1 frameshifting among different eukaryotic systems.
Studies of programmed -1 ribosomal frameshifting (-1 PRF) have been approached over the past two decades by many different laboratories using a diverse array of virus-derived frameshift signals in translational assay systems derived from a variety of sources. Though it is generally acknowledged that both absolute and relative -1 PRF efficiency can vary in an assay system-dependent manner, no methodical study of this phenomenon has been undertaken. To address this issue, a series of slippery site mutants of the SARS-associated coronavirus frameshift signal were systematically assayed in four different eukaryotic translational systems. HIV-1 promoted frameshifting was also compared between Escherichia coli and a human T-cell line expression systems. The results of these analyses highlight different aspects of each system, suggesting in general that (1) differences can be due to the assay systems themselves; (2) phylogenetic differences in ribosome structure can affect frameshifting efficiency; and (3) care must be taken to employ the closest phylogenetic match between a specific -1 PRF signal and the choice of translational assay system.
Base Sequence↗
Specific effects of ribosome-tethered molecular chaperones on programmed -1 ribosomal frameshifting.
The ribosome-associated molecular chaperone complexes RAC (Ssz1p/Zuo1p) and Ssb1p/Ssb2p expose a link between protein folding and translation. Disruption of the conserved nascent peptide-associated complex results in cell growth and translation fidelity defects. To better understand the consequences of deletion of either RAC or Ssb1p/2p, experiments relating to cell growth and programmed ribosomal frameshifting (PRF) were assayed. Genetic analyses revealed that deletion of Ssb1p/Ssb2p or of Ssz1p/Zuo1p resulted in specific inhibition of -1 PRF and defects in Killer virus maintenance, while no effects were observed on +1 PRF. These factors may provide a new set of targets to exploit against viruses that use -1 PRF. Quantitative measurements of growth profiles of isogenic wild-type and mutant cells showed that translational inhibitors exacerbate underlying growth defects in these mutants. Previous studies have identified -1 PRF signals in yeast chromosomal genes and have demonstrated an inverse relationship between -1 PRF efficiency and mRNA stability. Analysis of published DNA microarray experiments reveals conditions under which Ssb1, Ssb2, Ssz1, and Zuo1 transcript levels are regulated independently of those of genes encoding ribosomal proteins. Thus, the findings presented here suggest that these trans-acting factors could be used by cells to posttranscriptionally regulate gene expression through -1 PRF.
DNA-Binding Proteins↗
Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae.
5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semi-dominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.
Alleles↗
A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.
A wide range of RNA viruses use programmed -1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed -1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.
Animals↗
Torsional restraint: a new twist on frameshifting pseudoknots.
mRNA pseudoknots have a stimulatory function in programmed -1 ribosomal frameshifting (-1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for -1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168-174]. A separate 'torsional restraint' model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115-1127]. Here, experiments using a series of 'pseudo-pseudoknots' having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that -1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot.
Base Sequence↗
Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis.
There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed -1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5' (7)mGppp cap structure and 3' poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap(+)/poly(A)(+) and cap(-)/poly(A)(-) mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.
Alleles↗
Systematic analysis of bicistronic reporter assay data.
Bicistronic reporter assay systems have become a mainstay of molecular biology. While the assays themselves encompass a broad range of diverse and unrelated experimental protocols, the numerical data garnered from these experiments often have similar statistical properties. In general, a primary dataset measures the paired expression of two internally controlled reporter genes. The expression ratio of these two genes is then normalized to an external control reporter. The end result is a 'ratio of ratios' that is inherently sensitive to propagation of the error contributed by each of the respective numerical components. The statistical analysis of this data therefore requires careful handling in order to control for the propagation of error and its potentially misleading effects. A careful survey of the literature found no consistent method for the statistical analysis of data generated from these important and informative assay systems. In this report, we present a detailed statistical framework for the systematic analysis of data obtained from bicistronic reporter assay systems. Specifically, a dual luciferase reporter assay was employed to measure the efficiency of four programmed -1 frameshift signals. These frameshift signals originate from the L-A virus, the SARS-associated Coronavirus and computationally identified frameshift signals from two Saccharomyces cerevisiae genes. Furthermore, these statistical methods were applied to prove that the effects of anisomycin on programmed -1 frameshifting are statistically significant. A set of Microsoft Excel spreadsheets, which can be used as templates for data generated by dual reporter assay systems, and an online tutorial are available at our website (http://dinmanlab.umd.edu/statistics). These spreadsheets could be easily adapted to any bicistronic reporter assay system.
Data Interpretation, Statistical↗
Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough.
The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.
Adaptor Proteins, Signal Transducing↗
Ribosomal protein L3: influence on ribosome structure and function.
Early studies demonstrated roles for ribosomal protein L3 in peptidyltransferase center formation and the ability of cells to propagate viruses. More recent studies have linked these two processes via the effects of mutants and drugs on programmed -1 ribosomal frameshifting. Here, we show that mutant forms of L3 result in ribosomes having increased affinities for both aminoacyl- and peptidyl-tRNAs. These defects potentiate the effects of sparsomycin, which promotes increased aminoalcyl-tRNA binding at the P-site, while antagonizing the effects anisomycin, a drug that promotes decreased peptidyl-tRNA binding at the A-site. The changes in ribosome affinities for tRNAs also correlate with decreased peptidyltransferase activities of mutant ribosomes, and with decreased rates of cell growth and protein synthesis. In vivo dimethylsulfate (DMS) protection studies reveal that small changes in L3 primary sequence also have significant effects on rRNA structure as far away as 100 A, supporting an allosteric model of ribosome function.
Allosteric Site↗
A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element.
Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g. those containing frameshift mutations. Many viral mRNAs encode polycistronic messages where programmed -1 ribosomal frameshift (-1 PRF) signals direct ribosomes to synthesize polyproteins. A previous study, which identified consensus -1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted -1 PRF events would direct translating ribosomes to PTCs. Here we tested the hypothesis that a -1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral -1 PRF signal into a PGK1 reporter construct in the 'genomic' orientation. The results show that even low levels of -1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs. The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of -1 PRF frequencies can be used to post-transcriptionally regulate gene expression. Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the 'pioneer round' of translation in yeast.
Codon, Nonsense↗
Delayed rRNA processing results in significant ribosome biogenesis and functional defects.
mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed -1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at -1 frameshift signals, promoting increased programmed -1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis.
Alleles↗
The 9-A solution: how mRNA pseudoknots promote efficient programmed -1 ribosomal frameshifting.
There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed -1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 A by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5' direction, that is, a -1 ribosomal frameshift.
Bacteria↗
Decreased peptidyltransferase activity correlates with increased programmed -1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae.
Increased efficiencies of programmed -1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed -1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed -1 ribosomal frameshift signals.
Alleles↗
An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae.
A new in vivo assay system has been developed to study programmed frameshifting in the yeast Saccharomyces cerevisiae. Frameshift signals are inserted between the Renilla and firefly luciferase reporter genes contained in a yeast expression vector and the two activities are directly measured from cell lysates in one tube. Similar to other bicistronic reporter systems, this one allows the efficient estimation of recoding efficiency by comparison of the normalized activity ratios from each luciferase protein. The assay system has been applied to HIV-1 and L-A directed programmed -1 frameshifting and Ty1 and Ty3 directed +1 frameshifting. The assay system is amenable to high-throughput screening.
Animals↗