UC Irvine

Pre-mRNA splicing is required for the generation of functional mRNA transcripts. Splicing requires a large dynamic complex called the spliceosome to excise introns and ligate exons. This process requires accurate splice site selection. Various splice sites can be used within a gene to generate many different isoforms. Alternative pre-mRNA splicing is one of the most efficient systems to diversify the proteome. The ability to use different splice sites requires strict and fluid regulation to ensure the correct splicing combinations to safeguard against nonfunctional or deleterious protein isoforms. However this flexibility also permits the possibility of incorrect splice site selection leading to a loss of splicing fidelity.

Using next-generation sequencing on a three-exon min-gene to generate millions of spliced transcripts, we determined the splicing fidelity of each splice site down to the nucleotide level. We demonstrated that the 3’ splice site is more prone to splicing errors than the 5’ splice site. The rate at which the incorrect splice site was selected ranged between 1 in 631 and 1 in 131,611. Splice site recognition is highly reliant on the sequence of the mRNA. We demonstrated that single point mutations within the exon could have a drastic effect on splicing fidelity.

Using a bioinformatics approach, we examine the gene expression of splicing related genes in estrogen positive and estrogen negative breast cancer cell lines. We also surveyed the degree of alternative splicing in breast cancer, examining both between breast cancer and normal cell types and between different breast cancers. This analysis confirmed some known cancer related splicing programs and suggest the presence of new targets Using a splicing code we identified a generalized model suggesting that sequence conservation and some splicing regulators are the main components in cassette exon inclusion or exclusion.