Papers by Jonathan Dinman
Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mec... more Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10-R98S) found in T-ALL. The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10-R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10-R98S mice and T-ALL xenograft samples. RPL10-R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10-R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10-R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10-R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10-R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.
Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to p... more Programmed ribosomal frameshifting is a molecular mechanism that is used by many RNA viruses to produce Gag-Pol fusion proteins. The efficiency of these frameshift events determines the ratio of viral Gag to Gag-Pol proteins available for viral particle morphogenesis, and changes in ribosomal frameshift efficiencies can severely inhibit virus propagation. Since ribosomal frameshifting occurs during the elongation phase of protein translation, it is reasonable to hypothesize that agents that affect the different steps in this process may also have an impact on programmed ribosomal frameshifting. We examined the molecular mechanisms governing programmed ribosomal frameshifting by using two viruses of the yeast Saccharomyces cerevisiae. Here, we present evidence that pokeweed antiviral protein (PAP), a single-chain ribosomal inhibitory protein that depurinates an adenine residue in the ␣-sarcin loop of 25S rRNA and inhibits translocation, specifically inhibits Ty1-directed ؉1 ribosomal frameshifting in intact yeast cells and in an in vitro assay system. Using an in vivo assay for Ty1 retrotransposition, we show that PAP specifically inhibits Ty1 retrotransposition, suggesting that Ty1 viral particle morphogenesis is inhibited in infected cells. PAP does not affect programmed ؊1 ribosomal frameshift efficiencies, nor does it have a noticeable impact on the ability of cells to maintain the M 1 -dependent killer virus phenotype, suggesting that ؊1 ribosomal frameshifting does not occur after the peptidyltransferase reaction. These results provide the first evidence that PAP has viral RNA-specific effects in vivo which may be responsible for the mechanism of its antiviral activity.
has been paid to the reproductive biology of filarial nematode parasites as a possible target for... more has been paid to the reproductive biology of filarial nematode parasites as a possible target for immunological or chemotherapeutic
Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on eithe... more Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.
Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on eithe... more Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartate residues. This chemical cleavage is applied to the yeast ribosome proteome to evaluate its suitability for incorporation into high-throughput automated workflows. Peptide product mixtures were analyzed using either an HPLC-ESI-LTQ-Orbitrap or an HPLC-MALDI-TOF 2 . The peptides were readily identified, using MASCOT with a modified enzyme rule, and provided information about 73% of the proteome. Implications are considered of the extended length and the presence of multiple basic residues in these peptides.
Infectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae family, encodes a nonst... more 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-VP5 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 rNVI15infected 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.
rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs a... more rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome.
We report the cloning and characterization of a DNA damage-inducible (DDI) transcript DDI A121. T... more We report the cloning and characterization of a DNA damage-inducible (DDI) transcript DDI A121. The fulllength human DDI A121 cDNA contains an open reading frame of 113 amino acids, corresponding to a protein of 12.7 kDa. The deduced amino acid sequence of A121 shows high homology to the yeast translation initiation factor (eIF) sui1 and also exhibits perfect identity to the partial sequence of recently purified human eIF1. Expression of human A121 corrected the mutant sui1 phenotype in yeast, demonstrating that human A121 encodes a bona fide translation initiation factor that is equivalent to yeast sui1p. The mammalian A121/SUI1 gene exhibits two transcripts (1.35 kilobases and 0.65 kilobases) containing a common coding region but differing in their 3-untranslated region. The long and short A121/SUI1 mRNAs are differentially regulated by genotoxic and endoplasmic reticulum stress. The genotoxic stress induction of A121/SUI1 mRNA is conserved in both humans and rodents and occurs in a p53-independent manner. Our identification of a stress-inducible cDNA that encodes eIF1 suggests that modulation of translation initiation appears to occur during cellular stress and may represent an important adaptive response to genotoxic as well as endoplasmic reticulum stress.
Nematode spermatozoa, unlike their mammalian counterparts, arc nonflagellated crawling cells. The... more Nematode spermatozoa, unlike their mammalian counterparts, arc nonflagellated crawling cells. The pscudopod of these cells contains the major sperm protein (MSP) which comprises more than 15c~ of the protein in the sperm. MSP is presumed to function as a cytoskeletal element involved in motility. An Ascaris MSP eDNA sequence was used as a probe to identify and isolate Onchocerca volvulus MSP clones from a hgt 11 genomic library. Two clones, OVGS-1 (765 bp) and OVGS-2 (1765 bp), were charz,ctcrizcd by restriction endonuclease mapping and sequence analysis. Both gcnomic clones contain MSP protein coding regions of 99 and 282 bp separated by an intervening sequence of 153 bp. l'hc genes f)VGS-I and OVGS-2 arc 95% similar in nucleotide sequence in the protein coding regions, but only 79% similar in their intron sequences. A number of potential regulator) sequences in the flanking regions and at the exon,"intron junctions of the O. volvulus MSP genes arc in good agreement with consensus sequences in other cukaryotic cells. The nucleotidc sequence of the O. voh,ulus MSP genes wcrc over 80% similar to the Ascari.~ MSP eDNA sequence and 79% similar to the Caenorhabditis MSP-3 eDNA. The predicted amino acid sequence of the O. volvulus MSPs were 96% similar to each other, 90-91<~ similar to Ascaris MSP and 81-82% similar to Caenorhabditis MSP-3. These results offer evidence that the MSP sequences have been highly conserved throughout nematode evolution but are variable in their gcnomic organization and the presence of introns.
The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain prematu... more The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and͞or the process of programmed ؊1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed ؊1 frameshift efficiency. A upf3-⌬ strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed ؊1 ribosomal frameshift efficiency resulting in loss of the M 1 virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.
Background. Translating mRNA sequences into functional proteins is a fundamental process necessar... more Background. Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. This work investigates how ribosome structure and function are affected by rRNA base modification. The prevailing view is that rRNA base modifications serve to fine tune ribosome structure and function. Methodology/Principal Findings. To test this hypothesis, yeast strains deficient in rRNA modifications in the ribosomal peptidyltransferase center were monitored for changes in and translational fidelity. These studies revealed allele-specific sensitivity to translational inhibitors, changes in reading frame maintenance, nonsense suppression and aa-tRNA selection. Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay. rRNA chemical analyses of one of these mutants identified structural changes in five specific bases associated with the ribosomal A-site. Conclusions/Significance. Together, the data suggest that modification of these bases fine tune the structure of the A-site region of the large subunit so as to assure correct positioning of critical rRNA bases involved in aa-tRNA accommodation into the PTC, of the eEF-1ANaa-tRNANGTP ternary complex with the GTPase associated center, and of the aa-tRNA in the A-site. These findings represent a direct demonstration in support of the prevailing hypothesis that rRNA modifications serve to optimize rRNA structure for production of accurate and efficient ribosomes. Citation: Baxter-Roshek JL, Petrov AN, Dinman JD (2007) Optimization of Ribosome Structure and Function by rRNA Base Modification. PLoS ONE 2(1): e174.
High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homo... more High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 'P-site loop'. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.
Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central ... more Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunitjoining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented.
Yeast ribosomal proteins L11 and S18 form a dynamic intersubunit interaction called the B1b/c bri... more Yeast ribosomal proteins L11 and S18 form a dynamic intersubunit interaction called the B1b/c bridge. Recent high resolution images of the ribosome have enabled targeting of specific residues in this bridge to address how distantly separated regions within the large and small subunits of the ribosome communicate with each other. Mutations were generated in the L11 side of the B1b/c bridge with a particular focus on disrupting the opposing charge motifs that have previously been proposed to be involved in subunit ratcheting. Mutants had wide-ranging effects on cellular viability and translational fidelity, with the most pronounced phenotypes corresponding to amino acid changes resulting in alterations of local charge properties. Chemical protection studies of selected mutants revealed rRNA structural changes in both the large and small subunits. In the large subunit rRNA, structural changes mapped to Helices 39, 80, 82, 83, 84, and the peptidyltransferase center. In the small subunit rRNA, structural changes were identified in helices 30 and 42, located between S18 and the decoding center. The rRNA structural changes correlated with charge-specific alterations to the L11 side of the B1b/c bridge. These analyses underscore the importance of the opposing charge mechanism in mediating B1b/c bridge interactions and suggest an extensive network of information exchange between distinct regions of the large and small subunits.
To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions o... more To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is »2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and )2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.
Information must be shared and functions coordinated among the spatially distinct functional cent... more 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.
Programmed À1 ribosomal frameshifting (À1 PRF) is a mechanism that directs elongating ribosomes t... more Programmed À1 ribosomal frameshifting (À1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5 0 direction that is utilized by many RNA viruses. Importantly, rates of À1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, À1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of À1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor À1 PRF have evolved since the initial discovery of À1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of À1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of À1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.
To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions o... more To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is »2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and )2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.
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Papers by Jonathan Dinman