Jagjit Singh

Date of Award


Degree Type


Degree Name

Doctor of Philosophy in Regulatory Biology



Subject Headings

Molecular Biology


Nuclear precursor messenger RNA (Pre-mRNA) splicing is an important regulatory step in metazoan gene expression. More than 99% of nuclear pre-mRNA introns are U2-type that are spliced by U2-dependent spliceosome containing U1, U2, U4, U5 and U6 snRNAs. Only less than 1% of the introns are U12-type and spliced by U11, U12, U4atac, U5 and U6atac snRNAs. U12 and U6atac snRNAs play a central role in the splicing of U12-dependent introns. Our previous work demonstrated that the conserved 3' stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity, however any potential molecular mechanism was unclear. We discovered that the distal 3' stem-loop of U6atac has structural and sequence similarities with stem-loop III of U12 snRNA. These observations convinced us to investigate the structure-function requirement of the substructure of the U6atac 3' stem-loop in U12-dependent in vivo splicing. Our results show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3' stem-loop of U6atac. Using in vivo genetic suppressor assay, we demonstrate that stem-loop III of U12 snRNA which binds to p65 protein can be functionally replaced by U6atac distal stem-loop and vice-versa. Furthermore, we tested the compatibility of the U6atac 3' end from phylogenetically distant species in a human U6atac suppressor background to establish the evolutionary relatedness of these structures and in vivo functionality. In conclusion, we demonstrate that p65 C-terminal RNA recognition motif interacts with the U6atac distal 3' stem-loop. Although the significance of p65 binding to U6atac snRNA is not clear, our study suggests that both the helix structure, as well as the sequence of U6atac distal 3' stem-loop is important for snRNA-protein interactions and U12-dependent intron splicing.

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