Identification of RNA Regulatory Information in the Saccharomyces Cerevisiae Transcriptome PDF Download
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Author: Daniel Patrick Riordan
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Languages : en
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Book Description
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: Daniel Patrick Riordan
Publisher:
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Category :
Languages : en
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Book Description
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: Daniel Michael Klass
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Languages : en
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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: Nikoleta Georgieva Tsvetanova
Publisher:
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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: Torsten Hothorn
Publisher: CRC Press
ISBN: 1482204584
Category : Mathematics
Languages : en
Pages : 454
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Book Description
Like the best-selling first two editions, A Handbook of Statistical Analyses using R, Third Edition provides an up-to-date guide to data analysis using the R system for statistical computing. The book explains how to conduct a range of statistical analyses, from simple inference to recursive partitioning to cluster analysis. New to the Third Edition Three new chapters on quantile regression, missing values, and Bayesian inference Extra material in the logistic regression chapter that describes a regression model for ordered categorical response variables Additional exercises More detailed explanations of R code New section in each chapter summarizing the results of the analyses Updated version of the HSAUR package (HSAUR3), which includes some slides that can be used in introductory statistics courses Whether you’re a data analyst, scientist, or student, this handbook shows you how to easily use R to effectively evaluate your data. With numerous real-world examples, it emphasizes the practical application and interpretation of results.
Author: Jason Gabunilas
Publisher:
ISBN:
Category :
Languages : en
Pages : 175
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Book Description
RNA splicing is a critical component in the regulation of gene expression in all eukaryotes. The work described herein chronicles our investigative efforts into three facets of RNA splicing and their associated mechanisms in the model organism Saccharomyces cerevisiae. Previous work from our group highlighted the ability for nonsense-mediated mRNA decay (NMD) to mask the splicing defects of splicing factor mutants, suggesting that the full repertoire of splicing substrates and products is at least partly occluded by RNA surveillance mechanisms. Continuing this work, we sought to uncover previously unidentified splicing events by performing RNA-sequencing in wild-type yeast as well as strains deficient in NMD. This analysis revealed that alternative splicing at unannotated non-canonical 5'- and 3'-splice sites occurs within a large number pre-mRNAs in yeast, but that these events are not usually observed because they introduce premature termination codons (PTCs) into the translational reading frames of the spliced transcripts, thereby rendering them targets for degradation by NMD. This work demonstrated that the degree of alternative splicing in yeast RNA transcripts is greater than previously appreciated, and that alternative splicing linked to NMD (AS-NMD) serves to regulate overall transcript levels. Notably, this study uncovered a non-productive alternative 5'-splice site for the ribosomal protein gene RPL22B that is activated in a stress-dependent manner. We further investigated the splicing of RPL22B and found that its protein product Rpl22p functions in an extra-ribosomal capacity by inhibiting the splicing of its own pre-mRNA, defining an autoregulatory splicing-mediated negative feedback mechanism that fine-tunes the expression of Rpl22p with potential implications in stress response. Finally, we identified a global role for the second-step splicing factor Prp18p in the suppression of non-canonical alternative 3'-splice sites throughout the yeast transcriptome. Specifically, we found that branchpoint-proximal alternative 3'-splice sites are activated in the absence of Prp18p in a substantial fraction of intron-containing genes. These results suggest that Prp18p is responsible for maintaining the fidelity of RNA splicing. Together, these studies reveal new insights into gene regulation by highlighting the interplay between RNA splicing and quality control.
Author: Fabio Marchi
Publisher: BoD – Books on Demand
ISBN: 9535135031
Category : Medical
Languages : en
Pages : 330
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Book Description
The large potential of RNA sequencing and other "omics" techniques has contributed to the production of a huge amount of data pursuing to answer many different questions that surround the science's great unknowns. This book presents an overview about powerful and cost-efficient methods for a comprehensive analysis of RNA-Seq data, introducing and revising advanced concepts in data analysis using the most current algorithms. A holistic view about the entire context where transcriptome is inserted is also discussed here encompassing biological areas with remarkable technological advances in the study of systems biology, from microorganisms to precision medicine.
Author: Gregory A. Cary
Publisher:
ISBN:
Category :
Languages : en
Pages : 129
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Book Description
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.
Author: Stefan Hohmann
Publisher: Springer Science & Business Media
ISBN: 3540456112
Category : Science
Languages : en
Pages : 398
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Book Description
Every cell has developed mechanisms to respond to changes in its environment and to adapt its growth and metabolism to unfavorable conditions. The unicellular eukaryote yeast has long proven as a particularly useful model system for the analysis of cellular stress responses, and the completion of the yeast genome sequence has only added to its power This volume comprehensively reviews both the basic features of the yeast genral stress response and the specific adapations to different stress types (nutrient depletion, osmotic and heat shock as well as salt and oxidative stress). It includes the latest findings in the field and discusses the implications for the analysis of stress response mechanisms in higher eukaryotes as well.
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: Lesley J. Collins
Publisher: Springer Science & Business Media
ISBN: 1461403324
Category : Medical
Languages : en
Pages : 297
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Book Description
RNAs form complexes with proteins and other RNAs. The RNA‐infrastructure represents the spatiotemporal interaction of these proteins and RNAs in a cell‐wide network. RNA Infrastructure and Networks brings together these ideas to illustrate the scope of RNA‐based biology, and how connecting RNA mechanisms is a powerful tool to investigate regulatory pathways. This book is but a taste of the wide range of RNA‐based mechanisms that connect in the RNA infrastructure.
