Mechanism of Splicing of Unusual Intron in Human Proliferating Cell Nucleolar P120

The purpose of this project has been to gain a better understanding of pre-mRNA splicing mechanisms by studying SR proteins a family of trans-acting splicing factors, exonic splicing enhancers and non-conventional splicing of AT-AC introns. AT-AC introns are a rare type of intron present in a wide variety of genes including Pl2O. High Pl2O gene expression correlates with rapid cell proliferation as in cancer cells. The goals of the proposal have been met by work in three major areas of investigation. Specific consensus exonic splicing enhancers ESEs for individual SR proteins, 5F2ASF, SRp4O, SRp55 and 5C35, were identified by an in vitro SELEX procedure. Sequences matching these consensus ESEs stimulate splicing of conventional and non-conventional introns. A nonsense mutation in BRCAl, the breast cancer susceptibility gene, that is associated with familial breast and ovarian cancer causes skipping of the entire exon. This mutation is within a consensus SR protein binding site and in vitro splicing analysis showed that exon skipping is due to the failure of an SR protein, SE2ASF, to recognize the ESE. Using in vitro splicing complementation assays, SR proteins, which are required for conventional intron splicing, were shown to be required for excision of AT-AC introns.