Lattmann, Simon. Investigation of the guanine quadruplex resolving activity of the DEAH-box RNA helicase RHAU. 2012, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10316
In the first part of this research work, we undertook to address the molecular mechanisms underlying the specific recognition of G4 structures by RHAU. Through biochemical analysis of truncated and mutated recombinant forms of RHAU, we have uncovered the functional importance of the amino-terminal region for interaction with G4 structures and further identified within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. We also show that the G4-RNA substrate specificity and resolving activity shown by RHAU is an evolutionary conserved attribute in higher eukaryotes, insofar as CG9323, the Drosophila orthogolue of RHAU, binds and readily unwinds G4 structures.
In the second part of this work, we sought RNAs bound by RHAU in living cells. To this end, we employed high-throughput gene array technologies to identify RNAs associated with RHAU on a genome-wide scale. Approximately 100 RNAs were found to be significantly enriched with RHAU. Computational analysis of RNA sequences for potential intramolecular G4 structures revealed the preferential association of RHAU with transcripts bearing G4-forming motifs, suggesting direct targeting of G4-RNAs by RHAU. Among the most abundant RNAs selectively enriched, we identified the human telomerase RNA template TERC as a bona fide target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5'-region of TERC, both in vivo and in vitro. In-depth studies further revealed that RHAU was also part of the telomerase holoenzyme through direct interaction with TERC G4 structure. Collectively, these data provide the first evidence of a specific and direct interaction between a G4 resolvase enzyme and a potentially relevant intramolecular G4-RNA substrate, and more generally support the idea that intramolecular G4-RNAs are naturally occurring substrates of RHAU. Furthermore, these results provide circumstantial evidence for the existence of a G4-RNA structure in a fraction of the telomerase holoenzyme.
Overall, the present work brings new original insights regarding the mechanisms of G4 substrate recognition by RHAU and of its potential role as a G4 resolvase enzyme in vivo.
|Committee Members:||Gasser, Susan M. and Nagamine, Yoshikuni|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Growth and Development (Moroni)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||195 S.|
|Last Modified:||30 Jun 2016 10:52|
|Deposited On:||05 Mar 2013 11:23|
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