Date of Award

8-1-2015

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Gupta, Ramesh

Abstract

Stable cellular RNAs undergo a variety of post-transcriptional modifications, most of which are evolutionary conserved and found in functionally important regions of RNA. 2’-O-methylation of ribose sugars and conversion of uridine to pseudouridine by isomerization are the two most common modifications found in RNA in all the domains of life. 2’-O-methylation of the hydroxyl group of ribose is known to help in proper RNA folding, inhibit hydrolytic degradation and attack by nucleases on RNA. In Archaea, one of the ways this modification is known to be carried out is in an RNA-dependent manner by a Box C/D sRNP complex. This thesis sets to understand how exactly the RNA and proteins work together to bring about this modification. In the first part of this study, we aimed to investigate the role of Nop5, a protein component of the Box C/D sRNP whose function is not well understood. It was observed from previous work in our lab that aNop5p binds to single-stranded bulges and loops of some RNA. The binding seems to happen either by aNop5p alone or as a heterodimer with fibrillarin. This led us to hypothesize that Nop5 protein is responsible for binding to the target RNA to be methylated and bringing it to the assembling sRNP complex. To further investigate this idea, we identified two motifs in the C-terminal domain of the protein, the GAEK and ALFA motifs, mutated the motifs in the M. jannaschii versions of the protein and studied its significance in vitro using activity and binding assays. We have biochemically identified these motifs to be essential for the methylation activity of the sRNP complex and required to wedge open the guide strands for the target RNA to pair up. However, mutation of the motifs did not seem to change the way Nop5p binds to the bulges and loops of the RNA, inferring that these motifs are not essential for target identification and recruitment. The second part of the thesis is dedicated to the establishment and optimization of a pulldown technique using S1 RNA aptamer. This aptamer is inserted in a Box C/D guide RNA and used to purify the RNP complex formed in vivo using the strong affinity of the aptamer to streptavidin. The aptamer tag was successfully inserted into the 124mer form of sR-tMet of H. volcanii and the ability of the aptamer to bind to the streptavidin beads was tested. The roadblocks of the study, i.e. the non-specific binding and elution of the complex are currently being optimized.

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