Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in
splicing lead to human genetic disease, and splicing mutations in a number of genes involved in growth control
have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing
and splice site recognition are therefore fundamental to understanding regulated gene expression and human
disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the
splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of
trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate
the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue
culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure-
function and biochemical techniques. In the next five years, we aim to address several outstanding challenges
in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory
proteins display highly position-dependent activities that negatively or positively influence splice site choice
changed the way we think about the classical splicing activators (SR proteins) and the classical splicing
repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP
serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role to protect the
pre-mRNA from premature degradation. However, it is not understood how splicing regulators achieve
activation or repression of U1snRNP at the 5’ splice site. We aim to dissect the mechanisms of splicing
repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in
mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has
eluded extensive study. Thus, its regulation is not well-understood. The existence of inefficiently spliced
introns within coding exons (exitrons) further highlights the biological importance of understanding when
introns are removed efficiently and when they are not. We will decipher the rules of efficient intron removal
and investigate the impact of cis-acting elements in this process using synthetic biology approaches. The
argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater
than the testing landscape that the human genome offers. Here, we will take advantage of our expertise in
experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression
events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co-
transcriptionally. Defects in any one of these steps has been linked to disease. However, most published
studies evaluate only steady-state levels of gene expression or focus only on a single step. This ignores the
dynamics of gene expression steps that collectively contribute to the generation of proteins from mRNAs.
Thus, it is unclear how the kinetics of RNA processing and mRNA stability translate into an endpoint gene
expression signature. We have established a reliable method to metabolically label nascent RNA, which
allows us to track transcripts from synthesis to degradation. Work in this project will probe how steady state
mRNA levels are established, how the splicing and translation regulator SRSF1 influences mRNA dynamics
and how these processes adjust as a cell undergoes transformation. The goals of our research program are to
obtain a better understanding of exon recognition and alternative splicing. The new mechanistic insights will
be leveraged to improve strategies to therapeutically target this essential gene expression step.