Talk abstracts

Talk on Saturday 11:15-11:30am submitted by Jarnail Singh

Mechanism of splicing of large introns in human cells in vivo

Jarnail Singh (Department of Molecular Genetics, Cleveland Clinic, Cleveland, OH, 44195), Richard A Padgett (Department of Molecular Genetics, Cleveland Clinic, Cleveland, OH, 44195)

Abstract:
In mammalian genomes, majority of the genes (92%) contain introns. The average length of human introns is about 5.5kb, although human genome has more than 1200 introns larger than 100 kb and there are 9 introns that are larger than 500 kb in length. Large introns face a unique set of problems during splicing. For example, owing to their size, 5’ and 3’ splice sites might be far from each other spatially; the recognition of correct splice sites from the cryptic ones, since there are a number of cryptic exonic sequences present inside the large introns; and initial stability of 100s of kb long pre mRNA until it is spliced out. A number of potential mechanisms have been proposed to explain large intron splicing such as Recursive splicing which has been demonstrated in Drosophila, intra-splicing, intron skipping by RNA polymerase II or normal splicing. In this study, we used a number of different assays including use of transcription and splicing inhibitors, absolute quantitation of intronic sequences in the large introns, and shRNA dependent down-regulation of intron lariat debranching enzyme DBR1 to investigate the large intron splicing in human cells. Here we present evidence that the large introns are spliced in a normal fashion as their smaller counterparts in human cells and not by any other method as previously hypothesized.

Keywords: splicing, large introns