Student Theses and Dissertations

Duncan Smith

Date of Award

2009

Document Type

Thesis

RU Laboratory

Konarska Laboratory

Keywords

pre-mRNA splicing, splicing catalysis, spliceosome, S. cerevisiae, BS-U2 pairing

Abstract

Many details of the conformational dynamics underlying transitions during premRNA splicing, particularly during the catalytic phase, remain unclear despite our deep knowledge of spliceosome composition. The complex assembly phase that precedes catalysis, and the involvement of several motifs in multiple interactions along the splicing pathway, exacerbate the difficulty of accessing splicing catalysis experimentally. snRNAs likely form some or all of the spliceosomal active site. We attempted to establish an snRNA-only splicing system as a means to bypass assembly and recapitulate splicing catalysis. Our approach, however, yielded a 2’-3’ bondforming ribozyme activity in a linker region in our fused snRNA construct; this highlights the uncertainty inherent in minimised systems and, by contrast, the strength of experimental approaches based on complete spliceosomes. We describe the in vivo analysis of the interaction between the intron branch site (BS) and its cognate region of U2 snRNA; the absolute requirement for this basepairing interaction during spliceosome assembly has previously impeded the investigation of its role and fate during splicing catalysis. By substituting the entire duplex, we produced nonessential second-copy spliceosomes in which assembly and catalysis each partially limit the splicing reaction. Using such spliceosomes, we established unconfirmed details of branch site-U2 interaction, including a requirement for this duplex in bulging the branch nucleophile for first step splicing catalysis, and similar spatial organisation of key components of the spliceosome and group II self-splicing introns. Our second-copy spliceosomes should represent a system that can be extended to include other spliceosomal components, allowing the in vivo investigation of normally lethal, and even dominant lethal, alleles. In another line of experiments, we used RT-PCR to identify the products of reactions in which multiple sites in the BS-binding region of U2 were used in trans as 5’ splice sites during the splicing of a reporter gene. Characterisation of trans-splicing products revealed previously unknown dynamics in BS-U2 pairing, suggesting that this interaction may be disrupted between early assembly and first step catalysis, and again before the second step of the reaction. Our results also indicate a surprising degree of flexibility in the active site of the normally stringent S. cerevisiae spliceosome.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/429

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