RNA splicing is an essential process in the expression of genes in mammals. It is also frequently a point in regulation of gene expression via alternative splicing (AS) and through the retention of introns leading to degradation. As described below, recognition of signals for splicing such as 5'splice sites is critical for elongation by RNA polymerase and directionality of promoters. Further insights into these issues will advance the understanding of recently described long non-coding RNAs as well as many chronic, genetic, and infectious diseases. We recently found in analyzing RNA-seq data from our lab and other sources that a fraction of mammalian introns, about 6%, is more slowly spliced than their downstream introns in polyA + RNA. These "detained" nuclear introns are ultimately excised before the mRNA is transported to the cytoplasm. Analysis of splicing patterns indicates an enrichment of detained introns around AS cassette exons. However, a large fraction of detained introns is considered constitutively spliced, suggesting that removal of these introns could be a rate limiting regulatory step. We propose to complete documentation of detained introns as a subgroup and to study their roles in response to stress and other conditions. We have recently found that a well-studied RNA binding protein that regulates AS, Rbfox2, binds to conserved sites adjacent to cassette exons containing nonsense codons that are important in auto-regulation of RNA binding proteins (RBP). In these cases, Rbfox2 levels set a threshold for auto-regulation by the RBP allowing variation between cell states of these RBPs. The importance of this mechanism for cross regulation of different RNA binding proteins will be investigated. Both initiation and elongation of transcription are coupled to RNA splicing. This coupling likely occurs through chromatin modifications as well as through direct interactions of splicing complexes with polymerase. We reported the wide spread (over 70%) occurrence of divergent transcription from promoter sites in mammalian cells. We recently recognized that many long non-coding RNAs originate from divergent transcription. Preliminary evidence indicates that recognition of combinations of polyadenylation sites and 5'splice sites is important in controlling termination of transcription in the upstream antisense and sense direction. We propose to further investigate this relationship and the possibility that recognition of nascent upstream RNA can mediate regulation of transcription in the sense direction and chromatin modification.
The burden of cancer in public health is apparent in both human suffering and the cost of healthcare. The proposed research will provide the basis for new therapeutics to better treat cancer and thus improve public health.
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