Date Published: January 27, 2017
Publisher: Public Library of Science
Author(s): Joseph Russo, Jerome E. Lee, Carolina M. López, John Anderson, Thuy-mi P. Nguyen, Adam M. Heck, Jeffrey Wilusz, Carol J. Wilusz, Yoon Ki Kim.
We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion.
The concept of RNA regulons, in which functionally related genes are co-regulated by specific RNA-binding proteins (RBPs), was first proposed by Keene and Tenenbaum in 2002 . Since that time, the advent of RNA immunoprecipitation-based high throughput approaches including CLIP-seq  and PAR-CLIP  has facilitated the identification of large datasets of mRNA targets for a variety of RBPs, including CELF1 [4–8]. Additional studies have indicated that subcellular clustering of functionally related mRNAs through their interactions with RNA-binding proteins can result in a high local concentration of protein subunits to facilitate efficient macromolecular complex assembly . Based on these ideas, altered function of a single RBP could have profound effects on the assembly/abundance of an entire multi-protein complex. Such regulation could be central to normal cellular responses, but might also be detrimental if the RBP were mutated or impaired by disease.
In this study, we demonstrated that the CELF1 RNA-binding protein can specifically associate with sequences in the 3’UTR of each of the SRP mRNAs in vitro and in cultured myoblasts. Depletion of CELF1 resulted in stabilization of five SRP mRNAs and increased abundance of SRP19 and SRP68 proteins. Depletion of CELF1 also induced phenotypes consistent with disruption of the secretory pathway: CELF1 KD cells exhibited an increased ability to secrete a luciferase protein and slower migration in a wound healing assay. Finally, the defects in cell migration could be recapitulated by over-expression of SRP68, independent of CELF1 knockdown.