A multifaceted approach for discovering novel regulators of alternative splicing in Caenorhabditis elegans. Jennifer L Kabat

ISBN: 9780549660521

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NOOKstudy eTextbook

120 pages


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A multifaceted approach for discovering novel regulators of alternative splicing in Caenorhabditis elegans.  by  Jennifer L Kabat

A multifaceted approach for discovering novel regulators of alternative splicing in Caenorhabditis elegans. by Jennifer L Kabat
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Many alternative splicing events are regulated by intronic pentameric and hexameric sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selectiveMoreMany alternative splicing events are regulated by intronic pentameric and hexameric sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionarily conservation.

A WABA comparative alignment of C. elegans and C. briggsae identified 147 alternatively spliced genes that contain rare, compact, stretches of nucleotide conservation in introns surrounding alternative exons. Conserved intronic elements were collected into a database and algorithms were used to identify pentamers and hexamers present at higher levels in the conserved elements relative to total introns.

Many high scoring nematode motifs correspond to known mammalian splicing regulators, such as (T)GCATG, indicating the mechanism of alternative splicing regulation is well conserved in metazoans. This approach also identified novel sequences such as TCTATC, whose role in splicing is under investigation, allowing us to take a step forward in defining a catalog of splicing regulatory elements for an organism.

In vivo experiments testing two of the top scoring motifs, GCATG and TCTATC, demonstrated they were responsible for regulating alternative splicing of exons 15-19 of an unc-52 splicing reporter. When deleted individually, GCATG and TCTATC appear to exhibit opposing control on the splicing of exon 16. However, deletion of just these two motifs on either side of exon 16 has a unique splicing phenotype, suggesting these two sequences are just a small part of the larger system of splicing regulation for the unc-52 reporter.

Because many splicing factors function by binding short stretches of RNA (4-7 nucleotides) in a sequence-specific manner, I hypothesized that the sequence UCUAUC (the RNA sequence of the splicing regulatory motif TCTATC) could be used to identify the partner splicing factors for this novel motif. Using RNA affinity chromatography I demonstrated that the C. elegans homolog of human hnRNP R/Q, HRP-2, binds to the alternative splicing regulatory sequence UCUAUC.

Also discussed are ongoing experiments that attempt to link the function of the essential protein HRP-2 with alternative splicing regulation through UCUAUC elements.



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