The generation of distinct protein isoforms as a result of alternative pre-mRNA splicing is particularly prevalent in the immune system. Alternative splicing is reciprocally linked to signal transduction pathways: the majority of transcripts modulated by alternative splicing encode signaling proteins, and trans-acting factors that influence splicing decisions are induced or activated in response to signaling pathways. Alternative splicing of the highly abundant transmembrane protein tyrosine phosphatase, CD45, results in multiple isoforms that are expressed in a stage-specific manner during lymphocyte development. We identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as an inducible, critical regulator of activation- induced CD45 alternative splicing in a splicing factor RNA interference (RNAi) screen. HnRNPLL was necessary and sufficient to promote the transition from CD45RA to CD45RO isoform expression in a T cell line, B cell line and primary T cells. Global analysis of exon level expression revealed that hnRNPLL depletion resulted in alternative exon utilization in a number of additional immune-relevant transcripts, including CD44 and STAT5A. To better understand hnRNPLL activity with respect to CD45 as well as other transcripts, I will 1) explore the determinants of hnRNPLL binding at the molecular level, 2) identify the signaling cascade that culminates in induction of hnRNPLL and 3) modulate hnRNPLL expression in vivo to study its effect on lymphocyte development and function. I will additionally utilize the in vivo models to study the specific activities of the CD45 isoforms. My overall career goal is to spend one to two more year in a mentored phase in the laboratory of Anjana Rao, and then transition into my own laboratory. Anjana's laboratory is the ideal environment to initiate my aims. In addition to a rigorous academic environment, she has access to many cutting edge technologies that will facilitate the achievement of my goals. For instance, we have access to shRNA libraries for screening through the Broad Institute and a Solexa deep sequencer through the Harvard biopolymers facility. Anjana is also very involved in the scientific community and fosters many collaborations.
Given the broad distribution of alternative pre-mRNA splicing, characterization of the link between signaling and alternative splicing is highly significant, especially considering that 10-15% of human diseases are predicted to result from point mutations that affect alternative splicing. Knowledge of the mechanisms that support alternative splicing decisions may ultimately lead to novel therapies for this category of diseases.
