Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation

Alternative splicing is a critical component of human gene control that generates functional diversity from a limited genome. Defects in alternative splicing are associated with disease in humans. Alternative splicing is regulated developmentally and physiologically by the combinatorial actions of cis- and trans-acting factors, including RNA binding proteins that regulate splicing through sequence-specific interactions with pre-mRNAs. In T cells, the splicing regulator hnRNP L is an essential factor that regulates alternative splicing of physiologically important mRNAs, however the broader physical and functional impact of hnRNP L remains unknown. In this dissertation, I present analysis of hnRNP L-RNA interactions with CLIP-seq, which identifies transcriptome-wide binding sites and uncovers novel functional targets. I then use functional genomics studies to define pre-mRNA processing alterations induced by hnRNP L depletion, chief among which is cassette-type alternative splicing. Finally, I use integrative genomic analysis to identify a direct role for hnRNP L in repression of exon inclusion and an indirect role for enhancing exon inclusion that supports a novel regulatory interplay between hnRNP L and chromatin. In two appendices, I present CLIP-seq studies of two additional RNA binding proteins: the splicing factor CELF2 and the RNA helicase DDX17. In conclusion, I provide comparisons of these three CLIP-seq studies, providing high-level insights into the capabilities and limitations of CLIP-seq. In sum, this dissertation expands our knowledge of hnRNP L splicing control in the context of broader studies of RNA binding proteins in multiple cell types and conditions.