Poster abstracts

Poster number 75 submitted by Jagjit Singh

Splice site mutations alters the expression of intronic miRNA

Jagjit Singh (Dept of BGES, Cleveland State University), Neha Aggarwal (Dept of BGES, Cleveland State University), Kavleen Sikand (Dept of BGES, Cleveland State University), Girish C Shukla (Dept of BGES, Cleveland State University)

Abstract:
Two main classes of nuclear pre-mRNA introns, U2- and U12- types are found in metazoan genomes. Many U2-types introns codes for regulatory noncoding miRNAs. Existing in vitro studies show that processing of primary and precursor miRNA is independent of host pre-mRNA intron processing. Due to the nature of biochemical assays, these studies failed to observe a subtle variation if any, in mature miRNA production and its resultant effect on target expression. Numerous miRNA profiling studies have shown 2 to 4 fold up or down regulation, however if this differential expression exert similar effect on target gene expression is unclear. To test, if in vivo pre-mRNA processing has any subtle effect on mature miRNA production, we used a minigene containing four exons and three introns derived from MYH6 gene. The middle intron of the minigene codes for miR-208. The 5’ splice site of the miRNA containing intron was mutated from position 3 to 7. These mutants were cotransfected with a luciferase reporter containing 3’ UTR of THRAP-1, a validated target of miR-208. Our data show drastic in vivo splicing defect in positions 4 and 5 mutants. Real-time quantitative PCR of miR-208 indicates the lower level of miRNAs from these splicing defective mutant introns. Furthermore, luciferase reporter containing 3’ UTR of THRAP-1 expression was downregulated in 4 and 5 positions splice site mutants cotransfected experiments. This data suggest that splicing defective mutation affect miRNA production and its target gene expression.

Keywords: Splicing , intronic miRNAs