Download or read book Examination of the Coupling of Transcription to Pre mRNA Processing written by Barbara June Natalizio and published by . This book was released on 2008 with total page 276 pages. Available in PDF, EPUB and Kindle. Book excerpt: Eukaryotic precursor messenger RNA s (pre-mRNAs) are exclusively synthesized by RNA polymerase II (RNAP II) and require extensive processing for maturation into functional mRNAs. RNAP II transcription is coupled to pre-mRNA processing by mechanisms that are not well understood. In order to elucidate the mechanisms that mediate the functional coupling of transcription to pre-mRNA processing, we established an in vitro system capable of transcribing and splicing complex transcripts. Using this system, we observed that transcripts synthesized by RNAP II are spliced more efficiently than the same transcripts synthesized by T7 RNA polymerase (T7 RNAP). Furthermore, we demonstrated that this system could be used as a method to study alternative splicing.

Download or read book Investigating the Cotranscriptional Regulation of Pre mRNA Splicing and 3 end Processing microform written by Emanuel Rosonina and published by National Library of Canada = Bibliothèque nationale du Canada. This book was released on 2004 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt: Transcriptional activators play an important role in the assembly of the transcriptional apparatus at the promoter regions of genes. We examined whether activators participate in the coupling of transcription with pre-mRNA processing, as well. Strong activation domains resulted in higher levels of splicing and cleavage compared to weak activation domains when targeted to the promoter of reporter genes. Truncation of the CTD abrogated this effect indicating that the CTD is involved in mediating the effect of strong activators on efficient processing. Further exploration of this mechanism revealed that splicing factor PSF binds preferentially to strong activation domains, and stimulates splicing and cleavage in vivo, in a CTD dependent manner. Therefore, PSF likely mediates the effect of a strong activator on efficient processing, whereby strong activators facilitate the association of PSF with the elongation apparatus. Our findings therefore implicate both transcriptional activators and PSF in cotranscriptional splicing and 3 '-end formation. The production of a messenger RNA (mRNA) is a complex process that involves many concerted steps, including the processing of the primary transcript, or precursor mRNA (pre-mRNA). Processing involves capping, 3' -end cleavage and polyadenylation, and splicing of introns from within the pre-mRNA. Pre-mRNAs are transcribed by RNA polymerase II (pol II), and it has been found that pre-mRNA processing is coupled to transcription by pol II, facilitating efficient and coordinated production of mature mRNA. Here I report the results of investigations of cotranscriptional splicing and 3'-end formation of pre-mRNAs. The carboxyl-terminal domain (CTD) of pol II is a highly-repetitive sequence unique to pol II that plays key roles in coupling gene expression events leading to the production of mRNA. We examined the CTD requirement for processing of different pre-mRNAs by exploring the effect of CTD mutations on splicing and cleavage of reporter genes in mammalian cells. We found that the length, rather than the type of CTD repeats, can be the major determinant in the efficient processing of pre-mRNA substrates. Furthermore, our results suggest that the requirement for the CTD in pre-mRNA processing is dependent on sequences within the gene itself. The degree of CTD-dependence therefore appears to be pre-mRNA specific.

Download or read book Transcription and Splicing written by B. D. Hames and published by Oxford University Press, USA. This book was released on 1988 with total page 238 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis.

Download or read book Investigating the Connections Between Pre mRNA Splicing Chromatin Remodeling and Transcription and 3 end Processing written by Laura-Oana Albulescu and published by . This book was released on 2013 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt: Pre-mRNA splicing is an essential eukaryotic pathway which controls gene expression. Increasing lines of evidence indicate links between splicing and other RNA processing pathways such as chromatin remodeling, transcription and 3'end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. To determine the full complement of factors which impact pre-mRNA splicing, I developed a genome-wide screen in Saccharomyces cerevisiae which allowed me to evaluate differences in splicing efficiency in the background of ~5500 unique gene mutations. By measuring expression changes in precursor levels by high-throughput quantitative PCR, I detected enrichment in several classes of genes, with very strong candidates mapping to the chromatin remodeling, transcription and 3'end processing classes. One of these candidates is the bromodomain protein Bdf1, a component of the transcription factor TFIID and also a member of the SWR-C chromatin remodeling complex. Splicing sensitive microarrays confirm that deletion of Bdf1 leads to a global splicing defect, while ChIP-qPCR data reveal a decrease in U1 snRNP recruitment at intron containing genes, suggesting an inhibitory effect on spliceosome assembly. Conversely, Bdf1's homologue Bdf2 with which it is 46% identical, does not impact pre-mRNA splicing or spliceosome recruitment, consistent with my hypothesis that Bdf2 functions mainly in transcription. To further characterize Bdf1 function, I modified the high-throughput screening approach described above and employed it in a forward genetic manner to enable a mutagenic analysis of the Bdf1 protein. This analysis revealed that the C-terminal tail which overlaps with the Taf7 interaction domain, and contains a conserved SEED region and one of the known phosphorylation sites in Bdf1, may be responsible for the splicing defect. In opposition to the global splicing defect exhibited by Bdf1, mutations in 3'end processing factors such as Cft2 and Yth1 result in transcript-specific defects. My results highlight the cross-talk between 5' and 3'end processing factors and the spliceosome, and support a model in which the definition of terminal exons in the budding yeast is identical with the mechanism described in higher systems. Furthermore, the novel role of Bdf1 at the interface of transcription and pre-mRNA splicing suggests a new mechanism that underlies the coupling between these two RNA pathways.

Download or read book Spliceosomal Pre mRNA Splicing written by Klemens J. Hertel and published by Humana. This book was released on 2014-02-21 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Providing a guide to classical experimental approaches to decipher splicing mechanisms and experimental strategies that rely on novel multi-disciplinary approaches, Spliceosomal Pre-mRNA Splicing: Methods and Protocols describes the theory of alternative pre-mRNA splicing in seven introductory chapters and then introduces protocols and their theoretical background relevant for a variety of experimental research. These protocol chapters cover basic methods to detect splicing events, analyses of alternative pre-mRNA splicing in vitro and in vivo manipulation of splicing events and high-throughput and bioinformatic analyses of alternative splicing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, Spliceosomal Pre-mRNA Splicing: Methods and Protocols will aid newcomers and seasoned molecular biologists in understanding the fascinating world of alternative splicing with the ultimate goal of paving the way for many new discoveries to come.

Download or read book Evidence for Coupling Transcription and Splicing in Vivo in Saccharomyces Cerevisiae written by Luh Tung and published by . This book was released on 2007 with total page 191 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: The genetic information transfer pathway, which includes transcription, precursor messenger RNA (pre-mRNA) processing, mRNA export, translation, and mRNA turnover, is a highly integrated process in higher eukaryotes. This integration is mainly achieved by the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (Pol II), which serves as a platform for recruiting RNA processing factors. In yeast Saccharomyces cerevisiae, however, support for the coupling of transcription and splicing is far less assuring. Here we show, via our studies of Sub2p, a DExD/H-box protein essential for both splicing and mRNA export, that transcription and splicing are functionally coupled in yeast. The DExD/H-box proteins are often referred to as RNA helicases, but are increasingly viewed as ribonucleoprotein ATPases (RNPases) that can remodel specific RNPs in vivo. We first show that specific alterations of the intron branch-site binding protein (BBP) can eliminate the requirement for Sub2p, thus illuminating a principal role for Sub2p to remove BBP and Mud2p from the branch-site region. Unexpectedly, we uncovered a panoply of genetic and chemical perturbations of the yeast transcription machinery that can also bypass Sub2p's requirement. Chromatin-immunoprecipitation (ChIP) analysis revealed that these perturbations significantly reduce the effectiveness of co-transcriptional recruitment of BBP, thereby alleviating the lethal outcome of losing Sub2p. These findings also suggest a potential selective advantage for yeast to exploit modifications in a kinetically coupled upstream pathway to offset the catastrophic loss of key components in the downstream pathways.

Download or read book Study of the Coupling Between Transcription and MRNA Processing Utilizing a Novel Bcl x Mini gene written by Sean C. Devanney and published by . This book was released on 2007 with total page 110 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Bel family of genes are fundamental to the apoptotic mechanism. Bcl-x a member of this family, is alternatively spliced to create two main isoforms a long (Bcl-xL) and a short (Bcl-xS) variant. The long form exhibits anti-apoptotic activity, while the short form favors apoptosis. The proper balance of expression of these two isoforms is crucial for several developmental processes such as thymic selection and neural reshaping. A number of cancer types have been shown to over-express the long form, thereby granting them some protection from apoptosis. To study the transcriptional and post-transcriptional mechanisms regulating gene expression, the Bcl-x gene has been utilized. A complex mini-gene construct has been create in order to monitor the effects that promoter sequences, 5'UTR and 3'UTR's have on mRNA splicing, RNA export, stability and translation. Abundant evidence exists indicating that RNA processing events such as transcription, splicing and export are coupled, yet the mechanisms and factors involved in regulating these processes are poorly understood. The mini-gene is identical to the endogenous gene with the exception of a deletion to the 50Kb intron and the addition of a tag to differentiate the mini-gene product from the endogenous mRNA and protein. This novel system allows for the study of transcriptional and post-transcriptional mechanisms regulating gene expression from RNA biogenesis on to the protein level.

Download or read book Exploring the Mechanisms of Pre mRNA Splicing Using Mathematical Models of Coupled Transcription and Splicing written by Jeremy Davis-Turak and published by . This book was released on 2014 with total page 87 pages. Available in PDF, EPUB and Kindle. Book excerpt: One of the main challenges in modern biology is understanding how and when genes are turned on. As our knowledge of transcription regulation has matured, bioinformatic analyses have allowed increasingly quantitative predictions of gene expression. The ultimate goal of such analyses is to predict gene expression from concentrations of proteins in the cell, based on protein-DNA networks. Yet this is a highly ambitious task in Eukaryotes, since their gene expression is determined by chromatin conformation, epigenetic factors, promoter and enhancer states, rates of transcription initiation, elongation, transcript processing and termination, and mRNA export and stability. This thesis is focused on the co-occurrence of transcriptional elongation and pre-mRNA splicing, the process in which introns are removed from the pre-mRNA transcript. Splicing is an important regulatory step because aberrant splicing leads to either reduced or non-functional protein expression, and alternative splicing expands the repertoire of functional proteins encoded by the genome. The co-transcriptional nature of splicing implies that the kinetics of elongation in relation to splicing are important for the outcome of splicing decisions. Co-transcriptional splicing (CTS) has been extensively studied, but quantitative models of transcription networks that predict gene expression timecourses have yet to incorporate CTS considerations. Here I constructed kinetic models of CTS. Initially, I built a model of constitutive CTS and developed methods to fit nascent RNA-seq data to the model. Fitting this model to published datasets indicated that only a subset of genes can be expected to process all of their introns co-transcriptionally. Detailed data-mining of high-throughput datasets and genomes revealed patterns of compensatory signatures in sequence, chromatin and polymerase data, suggesting an evolutionary selection towards splicing co-transcriptionally. Next I expanded the model to include alternative splicing reactions. Despite the exponential combinatorial complexity, all possible isoforms resulting from up to nine introns can be simulated. A further expansion of the model considers separate reactions at the 5' and 3' ends of introns, which allows for simulation of phenomena such as exon definition and polymerase-mediated recruitment. Together, these novel tools can be used to test quantitative predictions of genome-wide splicing outcomes, or be incorporated into larger gene expression models.

Download or read book Eukaryotic MRNA Processing written by Adrian Krainer and published by IRL Press. This book was released on 1997 with total page 408 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume focuses on the major aspects of post-transcriptional mRNA processing in the nucleus of eukaryotic cells. Each of the described mRNA reactions is required for proper gene expression and can also serve as a control point for regulating the expression of many genes, for example duringembryonic development or in different cell types. The different chapters review the assembly of newly synthesized nuclear mRNA transcripts into hnRNP particles and catalytically active spliceosomes; the structure and mechanism of action of small nuclear ribonucleoprotein particles and proteinfactors that catalyse pre-mRNA splicing in mammalian cells and in yeast; the regulation of gene expression and generation of protein isoform diversity by alternative splicing; the mechanisms of 3' end cleavage and polyadenylation; the architecture of the cell nucleus in relation to these processesand to the localization of the relevant substrates and factors; the diverse mechanisms of RNA processing by ribozymes and their potential relevance for nuclear mRNA processing; the mechanism of spliced-leader addition by trans-splicing in nematodes and trypanosomes; and the process ofinsertion/deletion mRNA editing in kinetoplasmid protozoa. In each chapter, leading researchers have provided detailed, critical reviews of the history, experimental approaches, major advances, current ideas and models, as well as future directions, for each of these active areas of research.

Download or read book Nuclear pre mRNA Processing in Plants written by A. S. N. Reddy and published by Springer Science & Business Media. This book was released on 2008-04-16 with total page 323 pages. Available in PDF, EPUB and Kindle. Book excerpt: During the last few years, tremendous progress has been made in understanding various aspects of pre-mRNA processing. This book, with contributions from leading scientists in this area, summarizes recent advances in nuclear pre-mRNA processing in plants. It provides researchers in the field, as well as those in related areas, with an up-to-date and comprehensive, yet concise, overview of the current status and future potential of this research in understanding plant biology.

Download or read book Regulation of Alternative Splicing written by Philippe Jeanteur and published by Springer Science & Business Media. This book was released on 2002-10-21 with total page 272 pages. Available in PDF, EPUB and Kindle. Book excerpt: The discovery in 1977 that genes are split into exons and introns has done away with the one gene - one protein dogma. Indeed, the removal of introns from the primary RNA transcript is not necessarily straightforward since there may be optional pathways leading to different messenger RNAs and consequently to different proteins. Examples of such an alternative splicing mechanism cover all fields of biology. Moreover, there are plenty of occurrences where deviant splicing can have pathological effects. Despite the high number of specific cases of alternative splicing, it was not until recently that the generality and extent of this phenomenon was fully appreciated. A superficial reading of the preliminary sequence of the human genome published in 2001 led to the surprising, and even deceiving to many scientists, low number of genes (around 32,000) which contrasted with the much higher figure around 150,000 which was previously envisioned. Attempts to make a global assessment of the use of alternative splicing are recent and rely essentially on the comparison of genomic mRNA and EST sequences as reviewed by Thanaraj and Stamm in the first chapter of this volume. Most recent estimates suggest that 40-60% of human genes might be alternatively spliced, as opposed to about 22% for C. elegans.

Download or read book Molecular Biology of The Cell written by Bruce Alberts and published by . This book was released on 2002 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Anatomy of Gene Regulation written by Panagiotis A. Tsonis and published by Cambridge University Press. This book was released on 2003-01-13 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: Table of contents

Download or read book Pre mRNA Processing written by Angus I. Lamond and published by Springer. This book was released on 2014-08-23 with total page 221 pages. Available in PDF, EPUB and Kindle. Book excerpt: he past fifteen years have seen tremendous growth in our understanding of T the many post-transcriptional processing steps involved in producing func tional eukaryotic mRNA from primary gene transcripts (pre-mRNA). New processing reactions, such as splicing and RNA editing, have been discovered and detailed biochemical and genetic studies continue to yield important new insights into the reaction mechanisms and molecular interactions involved. It is now apparent that regulation of RNA processing plays a significant role in the control of gene expression and development. An increased understanding of RNA processing mechanisms has also proved to be of considerable clinical importance in the pathology of inherited disease and viral infection. This volume seeks to review the rapid progress being made in the study of how mRNA precursors are processed into mRNA and to convey the broad scope of the RNA field and its relevance to other areas of cell biology and medicine. Since one of the major themes of RNA processing is the recognition of specific RNA sequences and structures by protein factors, we begin with reviews of RNA-protein interactions. In chapter 1 David Lilley presents an overview of RNA structure and illustrates how the structural features of RNA molecules are exploited for specific recognition by protein, while in chapter 2 Maurice Swanson discusses the structure and function of the large family of hnRNP proteins that bind to pre-mRNA. The next four chapters focus on pre-mRNA splicing.

Download or read book mRNA Processing and Metabolism written by Daniel R. Schoenberg and published by Humana. This book was released on 2004-02-05 with total page 270 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cells possess a wealth of posttranscriptional control mechanisms that impact on every conceivable aspect of the life of an mRNA. These processes are intimately intertwined in an almost baroque manner, where promoter context influences the recruitment of splicing factors, where the majority of pre-mRNAs undergo alternative splicing, and where proteins deposited during nuclear processing impact distal cytoplasmic processing, translation, and decay. If there is a unifying theme to mRNA Processing and Metabolism: Methods and Protocols, it is that mRNA processing and metabolism are integrated processes. Many of the techniques used to study mRNA have been described in a previous volume of this series (RNA–Protein Interaction Protocols, Susan Haynes, ed.) and specialized methods journals. In selecting topics for mRNA Processing and Metabolism: Methods and Protocols, I sought input on new and novel techniques and approaches that build on this foundation using technological advances in microscopy, whole genome sequencing, microarrays, mass spectrometry, fluorescent detection methodologies, and RNA interference. I have tried not to bias this book toward any single model organism, and approaches described in the various chapters use yeast, Drosophila, Xenopus, mice, plants, and cultured mammalian cells.

Download or read book Macromolecular Protein Complexes II Structure and Function written by J. Robin Harris and published by Springer Nature. This book was released on 2020-01-14 with total page 657 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book follows on from Volume 83 in the SCBI series (“Macromolecular Protein Complexes”), and addresses several important topics (such as the Proteasome, Anaphase Promoting Complex, Ribosome and Apoptosome) that were not previously included, together with a number of additional exciting topics in this rapidly expanding field of study. Although the first SCBI Protein Complex book focused on soluble protein complexes, the second (Vol. 87)addressed Membrane Complexes, and the third (Vol. 88) put the spotlight on Viral Protein and Nucleoprotein Complexes, a number of membrane, virus and even fibrillar protein complexes have been be considered for inclusion in the present book. A further book is also under preparation that follows the same pattern, in an attempt to provide a thorough coverage of the subject. Chapter 9 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

Download or read book Alternative pre mRNA Splicing written by Stefan Stamm and published by John Wiley & Sons. This book was released on 2012-08-01 with total page 660 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book was written for graduate and medical students, as well as clinicians and postdoctoral researchers. It describes the theory of alternative pre-mRNA splicing in twelve introductory chapters and then introduces protocols and their theoretical background relevant for experimental research. These 43 practical chapters cover: Basic methods, Detection of splicing events, Analysis of alternative pre-mRNA splicing in vitro and in vivo, Manipulation of splicing events, and Bioinformatic analysis of alternative splicing. A theoretical introduction and practical guide for molecular biologists, geneticists,clinicians and every researcher interested in alternative splicing. Website: www.wiley-vch.de/home/splicing