Poster abstracts

Poster number 4 submitted by Neha Aggarwal

In vivo mutational analysis of U2-dependent intron containing microRNA

Neha Aggarwal (Biological, Geological and Environmental Sciences, CSU, Cleveland), Kavleen Sikand (Biological, Geological and Environmental Sciences, CSU, Cleveland), Girish Shukla (Biological, Geological and Environmental Sciences, CSU, Cleveland)

Abstract:
A large number of miRNA genes are encoded in U2-dependent spliceosomal introns of mammalian genes. Current evidence suggests that the 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 primary and precursor miRNA processing. Using in vivo and in vitro methods, we examined the splicing of á-myosin heavy chain intron 27 and processing of miR-208, a resident of intron 27. The 5' splice site mutations of intron 27 do not affect primary miRNA processing. Complete elimination of 5' splice site activated a cryptic splicing. Interestingly, conversion of the 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 the intron to U12-type, albeit without a consensus U12-type branch site sequence. To test if this intron is spliced by minor spliceosome, we mutated CC5/6GG of the 5' splice site, which is essential for U12-dependent splicing, as the sequence is recognized by U11 and U6atac snRNAs sequentially. Surprisingly, these mutations neither completely abolished the splicing nor activated any cryptic splicing. Co-expression of appropriate U6atac and U11 suppressors results support the notion that the intron might be spliced by a combination of major and minor spliceosomal snRNAs. All known miRNAs are harbored in U2-dependent introns only; we wanted to check if miRNA can process from a U12-dependent spliceosomal intron. To test this we cloned miR 208 in a U12-depedent intron and assayed it for in vivo splicing. To our surprise, the intron spliced in in vivo system and miR208 got processed from the intron as detected by in vitro splicing assay. We also mutated the 5’ splice site of intron 27 at positions 3’ to 7’ individually to check the effect of these mutations on splicing of U2-dependent intron containing miRNA. Experiments are being pursued to determine if the plasticity of splice sites is an evolutionary derived feature of introns which code for miRNA genes.

Keywords: Splicing, MicroRNA, miR processing