Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Ribonucleoprotein complexes are responsible for some of the post-transcriptional modifications of RNA that occur within the cell, including 2'-O-methylation and pseudouridylation. These modifications contribute, among other things, to RNA folding, inhibition of degradation, and general cellular viability. In this study, we identify residues within the proteins of these complexes that are important to the functioning of the Box C/D and Box H/ACA complexes. Candidates were selected based on previous work and mutant versions of the proteins were introduced in-vivo. Assays were done to determine the functionality of the mutant complex. This work is divided into three parts, focused on the three proteins investigated. The first part is concerned with Nop5, a protein in the Box C/D RNP complex. Nop5 is known to interact with all other proteins and RNAs in the complex, and is believed to serve a primarily structural role, aligning the other components. Mutagenesis study of suspected significant amino acids in this protein showed that it is difficult to disrupt the operation of Nop5 with single changes, but is possible with more extensive mutation. The second part concerns Fibrillarin, the catalytic protein of the Box C/D ribonucleoprotein complex. Previous mutagenesis work identified several important amino acids involved with AdoMet transfer and complex formation. The methylation ability of these mutant complexes were further examined in this work by confirming that the same modification, or lack thereof, occurred at a second rRNA position. The final part of this work is about Nop10, part of the Box H/ACA complex. This work is only preliminary, but begins the process of testing suspected essential amino acids in the structure.
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