Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Characterization of Arabidopsis thaliana SR protein genes: mutations, alternative splicing, and ESE selection(2007-06-07) edmonds, jason matthew; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA processing in eukaryotes is a highly complex process requiring numerous steps and factors that can play roles in the regulation of functional protein production. SR proteins are a well-defined family of splicing factors identified by a conserved RNA Recognition Motif (RRM) and carboxyl-terminal arginine/serine (RS) repeats. SR proteins are known to bind to mRNA precursors via Exonic Splicing Enhancers, and to recruit U2AF and the U1 snRNP to promote splicing. I have identified mutations in five Arabidopsis thaliana SR protein genes that result in altered phenotypes. Two (scl28-1 and srp31-1) result in embryonic lethal phenotypes, while three others (sc35-1, sr45-1, and srp30-1) result in viable and fertile plants with a range of phenotypes. I have also found that mutations in individual SR protein genes can effect the ability of a specific sequence to act as an ESE and hence affect splicing efficiency. Because 16 of the 20 Arabidopsis thaliana SR proteins themselves are alternatively spliced, I have looked for cross regulation using RT-PCR analysis of isoform accumulation in alternatively spliced SR protein genes. I found that SR proteins do, in fact, regulate the alternative splicing of gene targets and do so in both a gene and a tissue specific manner. In order to begin to fully understand the relationship between individual SR proteins it is essential to know when and where they are expressed throughout development. I have studied the expression pattern of 16 of the 20 SR proteins in the roots of wild-type plants as well as sc35-1, srp30-1, and sr45-1 mutants. I have identified both spatial and temporal expression patterns for these 16 proteins relative to specific tissues that compose the root.Item BIOINFORMATIC ANALYSIS OF THE FUNCTIONAL AND STRUCTURAL IMPLICATIONS OF ALTERNATIVE SPLICING.(2007-01-23) Melamud, Eugene; Moult, John; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In higher Eukaryotes, upon transcription of a gene, a complex set of reactions take place to remove fragments of a sequence (introns) from transcribed RNA. A large macro-molecular machine (the spliceosome) recognizes the ends of introns, brings ends into close proximity and catalyzes the splicing reaction. The selection of the location of the ends of introns (splice sites) determines the final message produced at the end of the process. In some cases, an alternative set of splice sites are chosen, and as a consequence different message is produced. This phenomenon is known as alternative splicing. It is now realized that nearly every Human gene undergoes alternative splicing, producing large variability in types and number of transcripts produced. In this thesis, we examine the functional and structural consequences of alternative splicing on proteins, we look into the mechanism of formation of complex splicing patterns, and examine the role of noise in the process.