Influence of Spliceosome Proteins on RNA Interactions in Pre-mRNA Splicing PDF Download
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Author: Karli Lipinski
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Pre-mRNA splicing is a fundamental process governing gene expression in eukaryotes and is orchestrated by the spliceosome, a dynamic ribonucleoprotein complex. Spliceosomes catalyze splicing in two steps and are composed of both small nuclear RNAs (snRNAs; U1, U2, U4, U5, U6) and numerous proteins. Dynamic interactions between snRNAs, proteins, and the pre-mRNA substrate occur during splicing which are central to regulating splice site recognition, catalysis, and fidelity. This thesis examines multiple facets of RNA-protein interactions within spliceosome complexes beginning with snRNA biogenesis, through formation of the spliceosome active site, and during the second step of chemistry, exon ligation. Functional mechanisms into splicing, such as selection of intron recognition sites, are examined. Function of the main catalytic component of the spliceosome, the U6 snRNA, is first explored. Transcription and post-transcriptional processing of U6 is unique among snRNAs and therefore may be required to generate a functional U6. Transcription by RNAP II instead of RNAP III produces a functional U6 molecule. Defects in stability, likely a result of incorrect post-transcriptional processing and binding of stabilizing proteins, result in changes to in vivo distributions of spliceosome sub particles called snRNPs. Transcription of U6 by RNAP II is useful for the incorporation of genetic tags for endogenous fluorescent labeling or purification. Additionally, new applications of endogenous fluorescent labeling techniques within the U4 snRNA are presented, paving the way for single molecule studies of snRNA dynamics and Brr2 helicase function during activation. Mango and MS2 tags are well tolerated in the yeast U4 snRNA and with several of the tagged U4 constructs also minimally impacting splicing activities. Future single molecule experiments will examine the timing of the U4 snRNA release from the spliceosome compared to the release of Prp3, a protein associated with U6 and U4 snRNAs. Finally, validation of a proposed novel splicing factor, Fyv6, and study of its influence on 3' SS represent major contributions to the field of splicing. Utilizing a new high resolution P complex structure of the spliceosome containing Fyv6 solved by Max Wilkinson, I examined multiple contacts of Fyv6 with other splicing factors, notably Prp22. Genetic studies in yeast show that interactions with the protein Syf1 and Prp22 are important for Fyv6 function. The absence of Fyv6 from spliceosomes results in transcriptome-wide splicing defects, largely due to changes in 3' SS usage. From these studies, Fyv6 can be added to the list of splicing factors that impact the second step of splicing and affect fidelity of 3' SS selection.
Author: Karli Lipinski
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Pre-mRNA splicing is a fundamental process governing gene expression in eukaryotes and is orchestrated by the spliceosome, a dynamic ribonucleoprotein complex. Spliceosomes catalyze splicing in two steps and are composed of both small nuclear RNAs (snRNAs; U1, U2, U4, U5, U6) and numerous proteins. Dynamic interactions between snRNAs, proteins, and the pre-mRNA substrate occur during splicing which are central to regulating splice site recognition, catalysis, and fidelity. This thesis examines multiple facets of RNA-protein interactions within spliceosome complexes beginning with snRNA biogenesis, through formation of the spliceosome active site, and during the second step of chemistry, exon ligation. Functional mechanisms into splicing, such as selection of intron recognition sites, are examined. Function of the main catalytic component of the spliceosome, the U6 snRNA, is first explored. Transcription and post-transcriptional processing of U6 is unique among snRNAs and therefore may be required to generate a functional U6. Transcription by RNAP II instead of RNAP III produces a functional U6 molecule. Defects in stability, likely a result of incorrect post-transcriptional processing and binding of stabilizing proteins, result in changes to in vivo distributions of spliceosome sub particles called snRNPs. Transcription of U6 by RNAP II is useful for the incorporation of genetic tags for endogenous fluorescent labeling or purification. Additionally, new applications of endogenous fluorescent labeling techniques within the U4 snRNA are presented, paving the way for single molecule studies of snRNA dynamics and Brr2 helicase function during activation. Mango and MS2 tags are well tolerated in the yeast U4 snRNA and with several of the tagged U4 constructs also minimally impacting splicing activities. Future single molecule experiments will examine the timing of the U4 snRNA release from the spliceosome compared to the release of Prp3, a protein associated with U6 and U4 snRNAs. Finally, validation of a proposed novel splicing factor, Fyv6, and study of its influence on 3' SS represent major contributions to the field of splicing. Utilizing a new high resolution P complex structure of the spliceosome containing Fyv6 solved by Max Wilkinson, I examined multiple contacts of Fyv6 with other splicing factors, notably Prp22. Genetic studies in yeast show that interactions with the protein Syf1 and Prp22 are important for Fyv6 function. The absence of Fyv6 from spliceosomes results in transcriptome-wide splicing defects, largely due to changes in 3' SS usage. From these studies, Fyv6 can be added to the list of splicing factors that impact the second step of splicing and affect fidelity of 3' SS selection.
Author: Deming Xu
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Nuclear pre-mRNA splicing proceeds in a protein-RNA complex, the spliceosome, via a two-step transesterification reaction. Five spliceosomal small nuclear RNAs; (snRNAs) are brought to the pre-mRNA substrate in a process known as spliceosome assembly. Extensive RNA-RNA interactions and conformational changes occur during spliceosome assembly and maturation. However, very little is known about how RNA conformational changes are coordinated between the two steps of the splicing reaction, and how recognition of the 3' splice site by other factors is connected to the function of U5 loop 1 prior to and following the first splicing step. I performed a genetic screen in order to identify factors that are important for the U2/U6 helix II interaction and/or the function of the 5' -end of U2 snRNA, on the basis of synthetic lethality with an 11nt nucleotide substitution in the 5'-end region of U2 snRNA that could also perturb the U2/U6 helix II interaction. Six Slt (synthetic lethal with U2 snRNA) factors were isolated in this screen. Slt11p and Slt22p are new splicing factors, while the remainder correspond to previously identified splicing factors, Slt15p/Prp17p, Slt16p/Smd3p, Slt17p/Slu7p and Slt21p/Prp8p. Slt22p is a large RNA-dependent ATPase, whose activity is preferentially stimulated by pre-annealed U2/U6 snRNAs. Biochemical and genetic analyses indicated that its function is associated with U2/U6 snRNA interaction and that it may unwind intermolecular helix II prior to the formation of the active spliceosome. Slt2lp/Prp8p has been shown to be required for both steps of the splicing reaction. The 'slt'21/'prp'8-21 mutation has been mapped to a domain in Prp8p that is important for the recognition of the polypyrimidine tract preceding the 3' splice site. It is synthetically lethal with mutations in only the U2 part of U2/U6 helix II. Consistent with the notion that the 5'-end of U2 snRNA plays a role in the second splicing step, I found that U2 mutations in this region are synthetically lethal with several second-step mutations, including 'slt15'/'prp17-100', ' slu4'/'prp17-2', 'slt17'/' slu7-100', and 'slu7-1'. Slt11p is a putative RNA-binding protein containing two Zn-finger motifs. Although 'SLT11' is essential for viability only at elevated temperatures, its product is required for the efficiency of the splicing reaction. In the absence of Slt11p, 'slt17'/'slu7-100' and mutations in specific regions of U2, U5 and U6 snRNAs become lethal. (Abstract shortened by UMI.).
Author: Adrian Krainer
Publisher: IRL Press
ISBN:
Category : Language Arts & Disciplines
Languages : en
Pages : 408
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Book Description
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.
Author: Zdravko Lorkovic
Publisher: CRC Press
ISBN: 149871336X
Category : Science
Languages : en
Pages : 174
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Book Description
Gene expression in eukaryotes is regulated at different levels, which need to be coordinated to implement the information in the genome. Now it is clear that post-transcriptional regulation of gene expression such as pre-mRNA splicing, mRNA transport, editing, turnover and translation are as important as the control of transcription. In all aspects
Author: James G. Umen
Publisher:
ISBN:
Category : Protein binding
Languages : en
Pages : 596
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Book Description
Author: B. D. Hames
Publisher: Oxford University Press, USA
ISBN:
Category : Music
Languages : en
Pages : 238
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Book Description
This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis.
Author: Susan R. Haynes
Publisher: Springer Science & Business Media
ISBN: 1592596762
Category : Science
Languages : en
Pages : 485
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Book Description
The molecular characterization of RNA and its interactions with proteins is an important and exciting area of current research. Organisms utilize a variety of RNA–protein interactions to regulate the expression of their genes. This is particularly true for eukaryotes, since newly synthesized messenger RNA must be extensively modified and transported to the cytoplasm before it can be used for protein synthesis. The realization that posttranscriptional processes are critical components of gene regulation has sparked an explosion of interest in both stable ribonucleoprotein (RNP) complexes and transient RNA–protein interactions. RNA is conformationally flexible and can adopt complex structures that provide diverse surfaces for interactions with proteins. The fact that short RNA molecules (aptamers; see Chapter 16) can be selected to bind many different types of molecules is evidence of the structural variability of RNA. RNA molecules are rarely entirely single- or double-stranded, but usually contain multiple short duplexes interrupted by single-stranded loops and bulges; in some RNAs, such as tRNAs, the short duplexes stack on each other. Further variability is generated by the presence of non-Watson-Crick base pairs, modified nucleotides, and more complex structures, such as pseudoknots and triple-strand interactions.
Author: Tupa Basu Roy
Publisher:
ISBN:
Category : Messenger RNA.
Languages : en
Pages : 79
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Book Description
Coding regions or exons of most human genes are interrupted by noncoding intervening regions or introns. Removal of nuclear precursor messenger RNA (pre-mRNA) introns by RNA splicing is an essential step in eukaryotic gene expression. Two types of nuclear pre-mRNA introns are known as U2-dependent or major type and U12-dependent or minor type. Nuclear pre-mRNA introns are removed by two distinct sets of ribonucleoprotein complexes or spliceosomes, which are formed by five small nuclear RNAs (snRNAs) for each spliceosome. U6atac and U12 snRNAs are central to U12-dependent spliceosome and play essential roles in the removal of U12-dependent introns. U6atac and U12 snRNAs bind to the 5' splice site and branch site, respectively of an U12-dependent intron. In addition, it has been predicted that, U6atac and U12 snRNAs interact inter-molecularly to form helix I structure, which appears to be an essential element of the minor spliceosome. We have been studying U6atac and U12 inter-molecular base-pairing interaction using an in vivo mutation suppression assay. In this study, we have characterized U6atac and U12 mediated helix I intermolecular interactions and have shown in vivo existence of the predicted structure. In addition, we have also identified a region of U6atac snRNA which appears to be a structural analog of U12 snRNA stem III element. This element is important for the function of U12 snRNA and functions by binding to a RNA binding 65K protein, which is unique to minor spliceosome. We show that, analogous stem-loop of U6atac snRNA also interacts with 65K - RNA binding protein. However, functional significance of this interaction remained unclear. In summation, we have characterized sequential and dynamic RNA-RNA interactions between U4atac-U6atac and U6atac-U12 snRNAs. Our data show that, extensive and obligatory RNA-RNA interactions are critical to the splicing of U12-dependent introns.
Author: Klemens J. Hertel
Publisher: Humana
ISBN: 9781627039796
Category : Medical
Languages : en
Pages : 0
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Book Description
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.
Author: Xiaoyi Raymond Gao
Publisher: Academic Press
ISBN: 0128167270
Category : Science
Languages : en
Pages : 386
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Book Description
Genetics and Genomics of Eye Disease: Advancing to Precision Medicine thoroughly examines the latest genomics methods for studying eye disease, including complex eye disorders associated with multiple genes. GWAS, WES, WGS, RNA-sequencing, and transcriptome analysis as employed in ocular genomics are discussed in-depth, as are genomics findings tied to early-onset glaucoma, strabismus, age-related macular degeneration, adult-onset glaucoma, diabetic retinopathy, keratoconus, and leber congenital amaurosis, among other diseases. Research and clinical specialists offer guidance on conducting preventative screenings and counseling patients, as well as the promise of machine learning, computational statistics and artificial intelligence in advancing ocular genomics research. - Offers thorough guidance on conducting genetic and genomic studies of eye disease - Examines the genetic basis of a wide range of complex eye diseases and single-gene and Mendelian disorders - Discusses the application of genetic testing and genetic risk prediction in eye disease diagnosis and patient counseling
