Nitric oxide as a novel regulator of alternative splicing

PROJECT SUMMARY/ABSTRACT Protein post-translational modification (PTM) and alternative splicing enable the limited genome of a cell to dynamically respond to environmental changes by diversifying its protein repertoire. Previous studies have revealed that environmental cues impact alternative splicing through numerous PTMs of RNA-binding proteins associated with the spliceosome. S-nitrosylation, the reversible covalent PTM of a protein cysteine residue by the gaseous signaling molecule nitric oxide (NO) to form an S-nitrosothiol (SNO)-modified protein, has been shown to alter protein function to play profound roles on cellular physiology, including the regulation of gene expression. Specifically, S-nitrosylation is known to directly regulate key transcription factors as well as to modify enzymes that alter the epigenome. Intriguingly, RNA-binding proteins of the heterogenous nuclear ribonucleoprotein (hnRNP) family, including the polypyrimidine tract-binding protein (PTB), are common in proteomic identifications of SNO-modified proteins under multiple conditions and in many cell types. Because PTB is a master regulator of alternative splicing, we are exploring the central hypothesis that NO enables a cell to dynamically regulate RNA splicing through SNO modification of PTB. Our laboratory recently mapped S-nitrosylation to a single cysteine residue in PTB. Dramatic alternations in gene expression when cells are exposed to NO are absent when this cysteine is mutated, strongly supporting a role for SNO in regulating the activity of PTB with transcriptome-wide implications. This proposal will interrogate these findings through three independent aims: Aim 1 will develop a bioinformatic pipeline based on RNA sequencing analyses to identify specific alternative transcripts regulated by SNO-PTB, including those that may have an outsized effect on global gene expression; Aim 2 will elucidate how SNO affects PTB conformation and its association with protein and RNA components of the spliceosome, offering a molecular mechanism for the influence of NO on alternative splicing; and Aim 3 entails the use of a conditional knock-in mutant mouse in which PTB cannot undergo the SNO modification, allowing determination of the physiological role of SNO-PTB by observing the consequences of dysregulated NO signaling. This project will advance our understanding of the role of NO signaling as a crucial mechanism of the cellular response to environmental cues through SNO-modification of a central regulator of alternative splicing, PTB. Heretofore, global effects of NO on cellular function have been attributed to widespread modification of proteins. The role of NO in regulating alternative splicing is previously unappreciated, and provides new perspectives on dynamic regulation of cellular function in health and disease. This study will thus define the role of NO in alternative splicing for the first time, potentially opening new areas of research.