The RNA binding protein KSRP negatively regulates utrophin A expression in skeletal muscle

Abstract

mRNA stability is a key factor in determining the expression pattern of many genes and typically involves sequences in the 3′UTR and RNA-binding proteins (RBPs). We have previously identified regions in utrophin A transcripts important to regulate their stability via a conserved AU-rich element (Chakkalakal JV et al, Nucl. Acid Res., 2008). The purpose of the present study was to assess the effects of the RBP KSRP on expression of utrophin in muscle. Our data demonstrate that shRNA-mediated knockdown of KSRP in myoblasts caused a 2-fold increase in the expression of a reporter construct containing the utrophin 3′UTR. Moreover, a 3-fold increase in endogenous utrophin transcript and protein levels was apparent upon KSRP suppression. To demonstrate a physical association between KSRP and utrophin mRNAs, we performed RNA-immunoprecipitation (RIP) analyses using C2C12 cell and TA muscle extracts and an anti-KSRP anitodoby. RIP analysis revealed that utrophin A mRNA was targeted by KSRP in vitro and in vivo. Furthermore, co-electroporation of murine TA muscle with sh-KSRP and the utrophin A 3′UTR reporter construct resulted in an elevation of reporter activity. This was accompanied by an increase in endogenous utrophin expression. Together, these data demonstrate that KSRP negatively regulates the stability of utrophin A mRNA via its 3′UTR. Supported by CIHR, AFM (France) and MDA (USA).