Single Molecule Studies Of Spliceosomal Snrnas U2-U6

<p>Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging</p> <p>from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5</p> <p>and U6) involved in substrate positioning and catalysis. It has been postulated, but never</p> <p>shown experimentally, that the U2-U6 complex adopts at least two conformations that</p> <p>reflect different activation states. We have used single-molecule fluorescence to probe the</p> <p>structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and</p> <p>mutants of highly conserved regions of U2-U6. Our data show the presence of at least</p> <p>three distinct conformations in equilibrium. The minimal folding pathway consists of a</p> <p>two-step process with an obligatory intermediate. The first step is strongly magnesium</p> <p>dependent, and we provide evidence suggesting that the second step corresponds to the</p> <p>formation of the genetically conserved helix IB. Site-specific mutations in the highly</p> <p>conserved AGC triad and the U80 base in U6 suggest that the observed conformational</p> <p>dynamics correlate with residues that have an important role in splicing. We also report</p> <p>the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a</p> <p>corresponding set of base triple interactions discovered in the recent published crystal</p> <p>structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base</p> <p>triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the</p> <p>ones found in crystal structure of the self-splicing group II intron given the extensive</p> <p>similarities between the spliceosome and the group II intron. Our data agree very well</p> <p>with the hypothesis. There are a large number of proteins in the spliceosome that play</p> <p>vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural</p> <p>rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing</p> <p>that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex.</p> <p>This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits</p> <p>the binding of Mg2+ to U2/U6.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2010

Author dc:creator

• Guo, Zhuojun

Contributors dc:contributor

• David Rueda

• single molecule
• splicing
• U2-U6
• Biophysics

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/163

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1162