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Fair, Benjamin Jung Pleiss, Jeffrey A. Soloway, Paul Lis, John T. Yu, Haiyuan 2018-10-23T13:35:53Z 2018-10-23T13:35:53Z 2018-08-30 ProQuest Submission ID: 10975 ProQuest Publication ID: 10844199 https://hdl.handle.net/1813/59753 https://doi.org/10.7298/X4RV0KXV 10489851 The identification of splice sites and catalytic removal of introns via the spliceosome is an essential and regulated component of eukaryotic gene expression. While ever-increasing numbers of human genetic diseases can be linked to defects in the splicing pathway, our molecular understanding of how these mutations disrupt this complex process remains incomplete. To identify mutations which impact the splicing pathway I have developed a series of targeted-sequencing based quantitative genetic screens in S. pombe, a yeast species which is genetically tractable yet retains features of complex splicing patterns that have been lost in the S. cerevisiae lineage. Through these screens I have isolated conditional alleles of spliceosome components and investigated the mechanism by which some of these mutations disrupt splicing. Furthermore, I have identified genes in other nuclear processes, such as transcription and chromatin remodeling, which contribute to splicing outcomes. These findings suggest splicing is largely co-transcriptional and suggest specific genes by which transcription is functionally linked to splicing. Finally, I describe a novel approach for enriching RNA-sequencing libraries for splicing-informative reads. This technique allows for precise quantitation and discovery of rare splice products, including splice intermediates, which are poorly captured in other RNA-sequencing methods. en_US screen Sequencing splicing Genetics Molecular biology Yeast Transcription IDENTIFICATION OF SPLICING PATHWAY MUTATIONS VIA TARGETED SEQUENCING dissertation or thesis application/pdf Biochemistry, Molecular and Cell Biology Cornell University Doctor of Philosophy Ph. D., Biochemistry, Molecular and Cell Biology https://hdl.handle.net/1813/59810 Publication Cornell Theses and Dissertations Fair, Benjamin Jung 5893a6ea-7af3-41d7-abc6-04bcd26ab5df open.access open.access <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="04698eb2-8b7c-4f1f-b579-6f733be266b2"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>en_US</Language> <Title>IDENTIFICATION OF SPLICING PATHWAY MUTATIONS VIA TARGETED SEQUENCING</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2018-08-30</PublicationDate> <DOI>https://doi.org/10.7298/X4RV0KXV</DOI> <Authors> <Author> <DisplayName>Fair, Benjamin Jung</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <OrgUnit /> </Publisher> </Publishers> <Keyword>screen</Keyword> <Keyword>Sequencing</Keyword> <Keyword>splicing</Keyword> <Keyword>Genetics</Keyword> <Keyword>Molecular biology</Keyword> <Keyword>Yeast</Keyword> <Keyword>Transcription</Keyword> <Abstract>The identification of splice sites and catalytic removal of introns via the spliceosome is an essential and regulated component of eukaryotic gene expression. While ever-increasing numbers of human genetic diseases can be linked to defects in the splicing pathway, our molecular understanding of how these mutations disrupt this complex process remains incomplete. To identify mutations which impact the splicing pathway I have developed a series of targeted-sequencing based quantitative genetic screens in S. pombe, a yeast species which is genetically tractable yet retains features of complex splicing patterns that have been lost in the S. cerevisiae lineage. Through these screens I have isolated conditional alleles of spliceosome components and investigated the mechanism by which some of these mutations disrupt splicing. Furthermore, I have identified genes in other nuclear processes, such as transcription and chromatin remodeling, which contribute to splicing outcomes. These findings suggest splicing is largely co-transcriptional and suggest specific genes by which transcription is functionally linked to splicing. Finally, I describe a novel approach for enriching RNA-sequencing libraries for splicing-informative reads. This technique allows for precise quantitation and discovery of rare splice products, including splice intermediates, which are poorly captured in other RNA-sequencing methods.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>