Biochemical and Genetic Characterization of Factors that Influence Branchsite Selection During Pre-mRNA Splicing in Yeast PDF Download
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Author: Tucker Joe Carrocci
Publisher:
ISBN:
Category :
Languages : en
Pages : 251
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RNA splicing plays many critical roles in eukaryotic gene expression, yet even 40 years after its discovery, the mechanism by which the spliceosome recognizes and removes introns is still poorly understood. In my thesis, I describe methods to produce RNAs for studies of the spliceosome and my work towards understanding an early step in splicing, branchsite selection. In my thesis, I discuss methods to label RNA fluorescently. I used MS2 fusion proteins tags to the label RNAs in living yeast using small molecule fluorophores. These tags allow for visualization of localized RNAs and demonstrate that organic fluorophores can be targets to RNAs in living yeast. I then discuss work performed in collaboration with Dr. Jiacui Xu on the development of aptamers that can bind fluorescent molecules directly, obviating the need for protein. Finally, I show that the 10DM24 deoxyribozyme can be used to prepare long, site-specifically modified fluorescent pre-mRNAs and demonstrate methods that can circumvent debranching by the splicing factor, Dbr1. In the second portion of my thesis, I discuss work on the role of splicing factors that contribute to branchsite choice. I explored the role of cancer causing mutations in a protein called SF3b1 using the yeast spliceosome. Mutations of SF3b1 result in changes in branchsite usage by the spliceosome. These mutations can impair and improve nonconsensus intron splicing, which we propose derives from a change in the conformation of the protein. I also explored differences between the human and yeast proteins and define regions that cannot be exchanged despite high sequence conservation. These differences explain the lack of efficacy of the spliceosome inhibitor, pladienolide B on the yeast spliceosome. Finally, I used smFRET to observe dynamics in the splicing factor Prp5, which ensures proper branchsite usage by the spliceosome. I showed that Prp5 primarily adopts an open state in the absence of ATP and RNA but transitions to a closed state upon binding. This work provides a strong foundation to define the mechanism of Prp5 proofreading of the branchsite. Overall, my work has significantly contributed to our understanding of spliceosome and the mechanisms of branchsite selection.
Author: Tucker Joe Carrocci
Publisher:
ISBN:
Category :
Languages : en
Pages : 251
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Book Description
RNA splicing plays many critical roles in eukaryotic gene expression, yet even 40 years after its discovery, the mechanism by which the spliceosome recognizes and removes introns is still poorly understood. In my thesis, I describe methods to produce RNAs for studies of the spliceosome and my work towards understanding an early step in splicing, branchsite selection. In my thesis, I discuss methods to label RNA fluorescently. I used MS2 fusion proteins tags to the label RNAs in living yeast using small molecule fluorophores. These tags allow for visualization of localized RNAs and demonstrate that organic fluorophores can be targets to RNAs in living yeast. I then discuss work performed in collaboration with Dr. Jiacui Xu on the development of aptamers that can bind fluorescent molecules directly, obviating the need for protein. Finally, I show that the 10DM24 deoxyribozyme can be used to prepare long, site-specifically modified fluorescent pre-mRNAs and demonstrate methods that can circumvent debranching by the splicing factor, Dbr1. In the second portion of my thesis, I discuss work on the role of splicing factors that contribute to branchsite choice. I explored the role of cancer causing mutations in a protein called SF3b1 using the yeast spliceosome. Mutations of SF3b1 result in changes in branchsite usage by the spliceosome. These mutations can impair and improve nonconsensus intron splicing, which we propose derives from a change in the conformation of the protein. I also explored differences between the human and yeast proteins and define regions that cannot be exchanged despite high sequence conservation. These differences explain the lack of efficacy of the spliceosome inhibitor, pladienolide B on the yeast spliceosome. Finally, I used smFRET to observe dynamics in the splicing factor Prp5, which ensures proper branchsite usage by the spliceosome. I showed that Prp5 primarily adopts an open state in the absence of ATP and RNA but transitions to a closed state upon binding. This work provides a strong foundation to define the mechanism of Prp5 proofreading of the branchsite. Overall, my work has significantly contributed to our understanding of spliceosome and the mechanisms of branchsite selection.
Author: Shawn Robert Lockhart
Publisher:
ISBN:
Category : RNA splicing
Languages : en
Pages : 320
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Author: Usha Vijayraghavan
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 370
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Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1168
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Author: Katherine Anne Senn
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Pre-mRNA splicing, the removal of noncoding introns to form a protein coding mRNA, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins organized into small nuclear ribonucleoproteins (snRNPs), catalyzes splicing in two steps. These steps are highly regulated to ensure correct mRNA isoform production and protein expression. Core components of the spliceosome as well as additional auxiliary factors tune splicing for accuracy and regulation of alternative splicing outcomes. Because of the complexity of splicing regulation, there are many open questions about the factors involved and the regulation of alternative splicing. This thesis addresses the functions of two yeast proteins involved in splicing regulation and an alternatively spliced gene in humans.In chapters 2 and 3, I discuss the novel yeast splicing factor Fyv6. Genetic and biochemical evidence supports a role for Fyv6 in the second step of splicing, exon ligation. A collaborator has determined the structure of a Fyv6-containing spliceosome by cryo-electron microscopy. Using this structure, we have used biochemical and transcriptomics experiments to elucidate how Fyv6 affects 3' splice site choice. Yeast splicing regulatory factor Mer1 interacts with introns and the U1 snRNP to enhance splicing of a subset of meiotically-regulated pre-mRNAs by an as-yet undetermined mechanism. With the goal of elucidating the mechanism, I recapitulated Mer1-dependent splicing in vitro and used colocalization single molecule microscopy to examine how Mer1 affects the dynamics of U1 binding to pre-mRNAs (Chapter 4). Our results indicate Mer1 promotes splicing without significantly altering the observed distributions of U1 binding events. This study lays the groundwork for future studies of splicing regulation by Mer1 and other factors. Finally, I studied the expression and alternative splicing of human glucose transporter family member GLUT8 (Chapter 5). Multiple alternatively spliced GLUT8 mRNA isoforms exist, but only some encode viable proteins. One that does not is upregulated in cancers, which reduces GLUT8 protein expression. Collaborators showed that based on its localization, inability to transport glucose, and release of a cleaved peptide, the main functional isoform of GLUT8 may be a metabolic sensor.
Author: Laura-Oana Albulescu
Publisher:
ISBN:
Category :
Languages : en
Pages : 320
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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.
Author: William Francis Mueller
Publisher:
ISBN: 9781303001017
Category :
Languages : en
Pages : 176
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Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using \emph{in vitro} splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures. Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa). Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.
Author: Jan Barciszewski
Publisher: Springer Science & Business Media
ISBN: 9780792358619
Category : Science
Languages : en
Pages : 718
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Book Description
RNA Biochemistry and Biotechnology describes various aspects of nucleic acid and protein structure, mainly RNA structure and proteins, interacting with specific RNA species. Papers deal with DNA protein interactions, telomerase, aminoacyl-tRNA synthetases, elongation factor Tu, DNA repair, RNA structure, NMR technology, RNA aptamer interaction of biological macromolecules with metal ions. Two papers deal with theoretical aspects of RNA structure production and computer modelling. Many papers describe the possibility of commercial application of RNA biotechnology. One article discusses the impact of direct democracy on basic science supporting biotechnology. Readership: Advanced graduate students, Ph.D. students and young scientists as well as specialists in the field.
Author: Robert Louis Last
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Author: Zbigniew Stanisław Warkocki
Publisher:
ISBN: 9783868444391
Category :
Languages : en
Pages : 161
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