Genomic profiling of alternative isoform expression and RNA stability during fibroblast quiescence

Abstract

<bold>Genomic profiling of alternative isoform expression and RNA stability during fibroblast quiescence</bold>

Quiescence, the reversible exit from the cell division cycle, is an important physiological process that is critical for processes such as fibroblast proliferation during the wound healing response, lymphocyte activation during the immune response, and the maintenance of progenitor cells during stem cell quiescence. Large-scale changes in gene expression occur upon induction into quiescence and many of these genes are involved in RNA processing. Although changes in RNA splicing, polyadenylation, and RNA decay could be important regulators of quiescence gene expression patterns, global changes in RNA processing during fibroblast quiescence are not well characterized. The goal of this study is to identify significant changes in gene expression, transcript architecture, and transcript stability between proliferating and quiescent fibroblasts in order to further define global and gene-specific contributors to quiescence phenotypes.

We started by measuring steady state changes in gene expression and isoform switching between proliferating and quiescent fibroblasts using high-throughput RNA sequencing. From this analysis we were able to identify a set of significantly differentially expressed isoforms between the two states. Since the majority of these isoforms were localized to the 3'UTR of transcripts, we created a polyadenylation site enriched sequencing library to increase our detection of 3'UTR isoforms. We identified ~300 genes with significant differences in 3'UTR isoform expression and selected the gene reversion-inducing-cysteine-rich (RECK) gene to study in more depth due to its involvement in extracellular matrix remodeling. Knockdown analysis of RECK showed that lowered expression of the short isoform, which is the dominant isoform during proliferation, results in decreased migration of fibroblasts. Additionally, we observed a global trend of longer 3'UTR isoform expression during quiescence that is not correlated to global changes in mRNA stability.

This study provides a rich catalogue of genes with significantly different isoform expression between proliferating and quiescent fibroblasts. We also give an example of how the characterization of isoform-specific function can identify genes such as RECK with prognostic potential in proliferative diseases such as cancer.