Characterization of Novel RNA-protein Regulatory Interactions in Saccharomyces Cerevisiae PDF Download
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Author: Nikoleta Georgieva Tsvetanova
Publisher:
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Languages : en
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The dynamic processes of a living cell depend on the coordinated temporal and spatial regulation of the many steps of gene expression. Transcription regulation is one control point of gene expression, and a gene can also be regulated post-transcriptionally, by RNA-binding proteins (RBPs). The biological significance of post-transcriptional regulation is especially evident in cases, where RBP binding controls the temporal precision of suppression and activation of important cellular stress responses. We developed a proteome-wide experimental approach for in vitro identification of novel RBPs and RNA-protein interactions in Saccharomyces cerevisiae. We found 12 novel RNA-binding proteins, the majority of which, surprisingly, are currently annotated as enzymes with roles in metabolic processes. We next used this proteomic approach to screen for proteins specifically interacting with the HAC1 RNA, which mediates activation of the yeast unfolded protein response (UPR). We found that HAC1 associated reproducibly with four small yeast GTPases, three of which are of the Ypt family of ras-GTPases. We further characterized one of them, the yeast Rab1 homolog Ypt1, and showed that Ypt1 interacted with unspliced HAC1 RNA only in the absence of ER stress. Selective Ypt1 depletion increased HAC1 RNA stability and expression, and also affected timely recovery from UPR. By developing and applying a novel proteomic approach for studying RNA-protein interactions, we established Ypt1 as an important regulator of HAC1 expression and UPR signaling. This unexpected protein-RNA interaction provides a biochemical mechanism for coordinating the key cellular processes of vesicle trafficking and ER homeostasis.
Author: Nikoleta Georgieva Tsvetanova
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
The dynamic processes of a living cell depend on the coordinated temporal and spatial regulation of the many steps of gene expression. Transcription regulation is one control point of gene expression, and a gene can also be regulated post-transcriptionally, by RNA-binding proteins (RBPs). The biological significance of post-transcriptional regulation is especially evident in cases, where RBP binding controls the temporal precision of suppression and activation of important cellular stress responses. We developed a proteome-wide experimental approach for in vitro identification of novel RBPs and RNA-protein interactions in Saccharomyces cerevisiae. We found 12 novel RNA-binding proteins, the majority of which, surprisingly, are currently annotated as enzymes with roles in metabolic processes. We next used this proteomic approach to screen for proteins specifically interacting with the HAC1 RNA, which mediates activation of the yeast unfolded protein response (UPR). We found that HAC1 associated reproducibly with four small yeast GTPases, three of which are of the Ypt family of ras-GTPases. We further characterized one of them, the yeast Rab1 homolog Ypt1, and showed that Ypt1 interacted with unspliced HAC1 RNA only in the absence of ER stress. Selective Ypt1 depletion increased HAC1 RNA stability and expression, and also affected timely recovery from UPR. By developing and applying a novel proteomic approach for studying RNA-protein interactions, we established Ypt1 as an important regulator of HAC1 expression and UPR signaling. This unexpected protein-RNA interaction provides a biochemical mechanism for coordinating the key cellular processes of vesicle trafficking and ER homeostasis.
Author: Kiyoshi Nagai
Publisher: Oxford University Press, USA
ISBN:
Category : Medical
Languages : en
Pages : 302
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Book Description
The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.
Author: Daniel Patrick Riordan
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Languages : en
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The unique post-transcriptional behavior of each mRNA is thought to be largely determined by features present in its molecular sequence, representing a type of RNA regulatory code. However, the details by which distinct regulatory outcomes are programmed into the sequences of different transcripts are mostly unknown. We set out to identify features of yeast mRNAs that influence their post-transcriptional fates. Using bioinformatic and in vitro selection approaches, we characterized several RNA recognition elements involved in mediating specific interactions with individual yeast RNA-binding proteins (RBPs). Most of the RNA elements we uncovered were associated with significant mRNA expression changes and were phylogenetically conserved in related yeasts, providing insights into the function and evolution of the corresponding interactions. We also analyzed RNA-protein interaction sites for the yeast Puf3 RBP by high-throughput sequencing under different growth conditions. These results provided high-resolution experimental evidence for Puf3 binding at consensus RNA elements in the transcriptome, and enabled detailed comparisons of individual interaction sites. Finally, we developed complementary methods for transcriptome-wide mapping of potential sites of RNA 2'-O-methylation. Application of these methods to the yeast transcriptome successfully recovered known sites of RNA modification and suggested that ribose methylation of functionally-related transcripts may occur and influence the regulation of endogenous yeast mRNAs. Overall, these results contribute to understanding of how RNA sequence features help to specify global differences in gene expression characteristics.
Author: Karen Renee Christie
Publisher:
ISBN:
Category :
Languages : en
Pages : 600
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Regulation of the process of transcriptional elongation is an important control mechanism in the expression of some genes. To fully understand this form of regulation will require better understanding of the functions of transcription elongation factors. The goal of this work was to characterize the transcription elongation factor TFIIS from Saccharomyces cerevisiae, originally called P37. I demonstrated that, like the mammalian TFIIS proteins, the yeast protein stimulates RNA polymerase II to cleave the nascent RNA transcript and to read-through an intrinsic block to elongation. Investigation of the protein-protein contacts between TFIIS and RNA polymerase II indicated that the carboxyl-terminal domain of the largest subunit, subunit four, and subunit seven of the polymerase are not required for TFIIS to promote cleavage and read-through by the polymerase. In addition the carboxyl-terminal half of the yeast TFIIS protein is sufficient for both of these in vitro activities. This result is consistent with the previous results demonstrating the carboxyl-terminus of mouse TFIIS was sufficient to activate RNA polymerase in vitro.
Author: Daniel Michael Klass
Publisher:
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Category :
Languages : en
Pages :
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We are on the threshold of a new era in our understanding of that fantastic feat of regulation at the core of life itself--gene expression. The rapid pace of new developments in genome-wide, high-throughput technologies has allowed us unprecedented access to observe multiple stages of the gene expression program for nearly the entire genome. This has revealed a widespread discordance between mRNA abundance and protein abundance for many genes whose expression changes in response to environmental stimuli, and a significant coordination of post-transcriptional regulation for specific sets of related mRNAs at the levels localization, translation, decay, and the noise in gene expression. Despite this evidence suggesting the existence of a coordinated regulatory framework that potentially affects the fate of every mRNA in the cell, our efforts to discern the underlying structure and regulatory themes are hindered by an incomplete understanding of RNA-protein interactions. To advance our comprehension of post-transcriptional regulation, we developed new tools to identify which proteins bind to RNA, which of those bind concurrently, which RNAs are bound by a given protein, and where each protein binds on each RNA. Using our proteomic tools we discovered hundreds unexpected RNA binding proteins, uncovered new RNA binding domains, identified widespread, concurrent binding with several RNA binding proteins, and inferred functional information from the simultaneous binding partners of several RNA binding proteins. We used our genomic, sequencing-based tools to systematically interrogate a large set of diverse RNA binding proteins and we discerned new themes from the resulting data. This revealed significant differences in function, localization, and regulation among the proteins encoded by the targets of a given RNA binding protein based on binding position. These results suggest that the functional consequences of the RBP-RNA interaction are determined not only by whether an mRNA is bound by an RBP but also by the position of the binding site within the mRNA and its relation to the other RBPs that bind the same mRNA. Overall, we found evidence of an extensive regulatory framework involving hundreds of RNA binding proteins, encompassing nearly the entire transcriptome, and extending our understanding of the RNA-protein interactions at the heart of post-transcriptional regulation.
Author: Catherine Elizabeth Willett
Publisher:
ISBN:
Category :
Languages : en
Pages : 366
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Author: 方瓊瑩
Publisher:
ISBN:
Category :
Languages : en
Pages : 156
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Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 270
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In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below. I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate. Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate inresponse to any stalled ribosome. I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.
Author: Ashwini R. Jambhekar
Publisher:
ISBN:
Category :
Languages : en
Pages : 314
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RNA localization is an important regulatory mechanism utilized by many cells and organisms for ensuring proper development. Although this process has been studied for many years in Drosophila and Xenopus, the single-celled yeast S. cerevisiae also provides examples of RNA transport. A core complex of 3 proteins---She2, She3, and Myo4---transports ASH1 and IST2 mRNAs to bud tips of growing cells by an actinmyosin based mechanism. In this work, 22 additional mRNAs have been identified as She-complex targets and have been shown to localize to bud tips in vivo. In contrast to other organisms, S. cerevisiae was found to contain localization elements in the coding regions of transported RNAs. Unbiased selection of localization elements from the open reading frames of several She-complex targets revealed a short single-stranded RNA motif containing a core CG dinucleotide which was essential for She2/3 binding and transport. Further analysis also revealed additional context-dependencies affecting the function of the motif. These results establish yeast as a model system for studying RNA transport, and provide a foundation for elucidating the mechanisms of motor-cargo interactions and defining the range of regulatory functions fulfilled by localization of mRNA. Furthermore, the identification of a consensus motif and accessory features regulating She-complex recognition suggests a general mechanism by which mRNAs can simultaneously mediate protein production and binding of regulatory factors.
Author: Gregory A. Cary
Publisher:
ISBN:
Category :
Languages : en
Pages : 129
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Although gene expression begins with transcription, there are a variety of mechanisms that cells use to control and tune expression post-transcriptionally. Many post-transcriptional regulatory functions including translational regulation, transcript surveillance, intracellular RNA localization, and RNA decay occur in organelles known as RNA granules. RNA granules, such as processing (P)-bodies, are cytoplasmic accumulations of translationally repressed mRNA and associated proteins that are ubiquitous among eukaryotes. Much of what is known about RNA granule biology has been observed through genetic and cytological experimentation and very few biochemical enrichments of these structures have been reported. In this work I present an affinity enrichment strategy for Dhh1, a conserved core component of P-bodies, from the budding yeast Saccharomyces cerevisiae. We identify proteins co-enriching with Dhh1 using tandem mass spectrometry and show that many known RNA granule proteins are enriched by this approach. We go on to compare the association of proteins with the complex across two environmental conditions to examine the effect of stress induction on RNA granule assemblies. We find that metabolic enzymes and molecular chaperones are typically more abundant in the stress-induced P-body complex and demonstrate that one chaperone, YDJ1, is involved in the stress-induced aggregation of several P-body proteins into cytoplasmic foci. We also identify RNA co-enriching with Dhh1 and detect several classes of catalytic RNA as well as a strong enrichment for the mRNA encoding the P-body protein PAT1. Finally, I present and discuss the characterization of a yeast strain that exhibits sensitivity to the drug puromycin. The puromycin-sensitive strain incorporates the drug into nascent proteins in vivo and I discuss how this is a unique and useful approach for the detection of protein biosynthesis. The techniques developed and employed in this dissertation provide novel perspectives on post-transcriptional regulatory processes and enable further investigations into how these regulatory programs are executed within the cell.
