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||Size||Format||Hahn2011Supplementary Table S1.xlsx||one year restriction||961.67 kB||Microsoft Excel||Hahn2011.docx||one year restriction||20.93 MB||Microsoft Word||Hahn2011.pdf||one year restriction||14.9 MB||Adobe PDF|
|Title: ||Brr2 RNA helicase and its protein and RNA interactions|
|Authors: ||Hahn, Daniela|
|Supervisor(s): ||Tollervey, David|
|Issue Date: ||24-Nov-2011|
|Publisher: ||The University of Edinburgh|
|Abstract: ||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.|
|Sponsor(s): ||Darwin Trust of Edinburgh|
|Appears in Collections:||Biological Sciences thesis and dissertation collection|
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