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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Identification and characterization of small molecule inhibitors of pre-mRNA splicing that block spliceosome assembly at novel stages

Sidarovich, Anzhalika 17 April 2015 (has links)
No description available.
2

Brr2 RNA helicase and its protein and RNA interactions

Hahn, Daniela January 2011 (has links)
The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.
3

A new role for the spliceosome in the regulation of gene expression

Volanakis, Adam January 2012 (has links)
Through a genome wide study of spliceosome recruitment in Saccharomyces cerevisiae, we were able to identify a set of protein-coding genes that despite the fact that they contain no introns and their mRNA is not known to be spliced; their loci were occupied by the spliceosome. Bioinformatic analysis revealed the existence of splicing signals on these genes. Detailed analysis of BDF2, a representative gene, revealed that the spliceosome negatively regulates its mRNA levels through an unconventional one-step splicing reaction that cleaves BDF2 mRNA and targets the cleavage products for degradation. In an effort to clarify the mechanism of spliceosome recruitment to BDF2 locus, we identified that Bdf1, the redundant to Bdf2 factor, is required for the recruitment of the spliceosome at BDF2 and the subsequent down-regulation of its mRNA levels. The above led us to propose a new role for the spliceosome in the regulation of gene expression. Finally, we investigated the generality of this regulatory mechanism is S. cerevisiae and identified a set of genes which can be differentially spliced and whose physiological expression could be potentially regulated by the spliceosome.

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