Date of Award

2010

Degree Type

Thesis

Department

Biological, Geological and Environmental Sciences

First Advisor

Shukla, Girish

Subject Headings

MicroRNA processing, Messenger RNA, RNA splicing, Necleoproteins, miRNA, Intron, MicroRNA processing, Nuclear pre-mRNA splicing, miRNA, Intron

Abstract

Nuclear pre-mRNA splicing is an important step in eukaryotic RNA processing and can yield a variety of transcripts. Further, it is known that nuclear pre-mRNA introns are of two types, U2-dependent and U12-dependent. Recently discovered, microRNAs (miRNA) are short, endogenous, 20-22 nucleotide long, non-coding RNA molecules that base pair with target mRNAs to modulate translation. Current evidence suggests that processing of intronic miRNA does not affect the splicing. Since, intronic miRNAs are involved in essential cellular processes, we surmised if splice sites of miRNA coding introns are flexible to support productive precursor and mature miRNA processing. We constructed a minigene reporter consisting exons 26-29 including introns from MYH6 gene and an intronic miRNA, miR-208 harbored in intron 27. Using in vivo and in vitro methods, we examined splicing of MYH6 intron 27 and processing of miR-208. The predicted stem-loop structure of miR-208 and the 5' splice site of the intron 27 were mutated. Stem-loop mutations had no recordable splicing defect of intron 27. The 5'SS mutant activated a cryptic splicing event using a sub-optimal splice site located 50 nucleotides upstream of wild-type splice site. Interestingly, conversion of U2-type 5' splice site to consensus U12-type 5' splice site did not affect in vivo splicing of the intron, suggesting potential conversion of intron to U12-type, albeit without a consensus U12-type branch site sequence. However, miRNA processing was affected as established by in vitro splicing assay and qRT-PCR analysis of miR-208. We mutated nucleotides from position 3 to 7 in intron 27 to study if changes in splice site will allow miRNA processing to occur and if splicing will also be affected. Our data indicates significant changes in spliced phenotype as well as in miR-208 expression level. Since, all known intronic miRNAs are harbored in U2-dependent introns of mammalian genes, we wanted to study if miRNA processing will be compatible with U12-dependent intron. With this aim, w

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