Date of Award

2011

Degree Type

Thesis

Department

Biological, Geological and Environmental Sciences

First Advisor

Shukla, Girish

Subject Headings

Messenger RNA, RNA splicing, Nucleoproteins, RNA-RNA interactions, 65K-C-RRM protein, nuclear pre-mRNA splicing, U12-type or minor splicing, U6atac snRNA

Abstract

Coding regions or exons of most human genes are interrupted by noncoding intervening regions or introns. Removal of nuclear precursor messenger RNA (pre-mRNA) introns by RNA splicing is an essential step in eukaryotic gene expression. Two types of nuclear pre-mRNA introns are known as U2-dependent or major type and U12-dependent or minor type. Nuclear pre-mRNA introns are removed by two distinct sets of ribonucleoprotein complexes or spliceosomes, which are formed by five small nuclear RNAs (snRNAs) for each spliceosome. U6atac and U12 snRNAs are central to U12-dependent spliceosome and play essential roles in the removal of U12-dependent introns. U6atac and U12 snRNAs bind to the 5' splice site and branch site, respectively of an U12-dependent intron. In addition, it has been predicted that, U6atac and U12 snRNAs interact inter-molecularly to form helix I structure, which appears to be an essential element of the minor spliceosome. We have been studying U6atac and U12 inter-molecular base-pairing interaction using an in vivo mutation suppression assay. In this study, we have characterized U6atac and U12 mediated helix I intermolecular interactions and have shown in vivo existence of the predicted structure. In addition, we have also identified a region of U6atac snRNA which appears to be a structural analog of U12 snRNA stem III element. This element is important for the function of U12 snRNA and functions by binding to a RNA binding 65K protein, which is unique to minor spliceosome. We show that, analogous stem-loop of U6atac snRNA also interacts with 65K - RNA binding protein. However, functional significance of this interaction remained unclear. In summation, we have characterized sequential and dynamic RNA-RNA interactions between U4atac-U6atac and U6atac-U12 snRNAs. Our data show that, extensive and obligatory RNA-RNA interactions are critical to the splicing of U12-dependent introns

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