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Alternative splicing of pre-mRNA is a critical mechanism for enabling genetic diversity, and is a carefully regulated process in neuronal differentiation. RNA binding proteins (RBPs) are developmentally expressed and physically interact with RNA to drive specific splicing changes. This work tests the hypothesis that RBP-RNA interactions are critical for regulating timed and coordinated alternative splicing changes during neurodevelopment and that these splicing changes are in turn part of major regulatory mechanisms that underlie morphological and functional maturation of neurons. I describe our efforts to identify functional RBP-RNA interactions, including the identification of previously unobserved splicing events, and explore the combinatorial roles of multiple brain-specific RBPs during development. Using integrative modeling that combines multiple sources of data, we find hundreds of regulated splicing events for each of RBFOX, NOVA, PTBP, and MBNL. In the neurodevelopmental context, we find that the proteins control different sets of exons, with RBFOX, NOVA, and PTBP regulating early splicing changes and MBNL largely regulating later splicing changes. These findings additionally led to the observation that CNS and sensory neurons express a variety of different RBP programs, with many sensory neurons expressing a less mature splicing pattern than CNS neurons. We also establish a foundation for further exploration of neurodevelopmental splicing, by investigating the regulation of previously unobserved splicing events.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Turlough M. Finan.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 56-59). Also available for download via the World Wide Web; free to University of Oregon users.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA. / text

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<p>Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes.<br /> <br /> Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves.</p>

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-80). Also available for download via the World Wide Web; free to University of Oregon users.

Diss. (sammanfattning) Stockholm : Univ., 2004. / Härtill 4 uppsatser.