Murine Spam1 mRNA:: Involvement of AU-rich elements in the 3′UTR and antisense RNA in its tight post-transcriptional regulation in spermatids

Sperm adhesion molecule1 (SPAM1), the best characterized hyaluronidase gene, is abundantly expressed in the testis. We attempted to overexpress mouse Spam1 via transgenesis using either the endogenous promoter in a BAC or a heterologous Protamine1 promoter for a Spam1 cDNA transgene. Although transgene-copy numbers ranged from 2 to 15 and transgenic transcripts were expressed, there was a general failure of overexpression of the RNA and protein in the testis of all seven founders. Also, three transgenic lines showed a modest down-regulation or co-suppression of the RNA for Spam1 and Hyal5, present on the BAC. We provide evidence for the potential involvement of two co-ordinating post-transcriptional regulatory processes in the failure of overexpression: abundant endogenous antisense RNA and adenosine-uridine (AU)-rich element-mediated regulation of RNA turnover. We demonstrate that AU-rich elements (AREs) in the 3'UTR of mRNAs, well-known to interact with trans-acting proteins to target the RNA for (in)stability, are present in Spam1 RNA and specifically bind to six testicular cytoplasmic proteins. These AU-binding proteins (AUBPs) were virtually absent from the kidney where transcripts are rare, and were shown to interact with the cytoskeleton, which modulates mRNA turnover. In addition to a role in the RNAi pathway, antisense RNA can also modulate ARE-mediated regulation of mRNA by hybridizing to the AREs and specifically silencing their function. This potentially links the two processes in the regulation of Spam1 expression. We hypothesize that testicular Spam1 RNA is regulated post-transcriptionally by cis-acting ARE(s) in the 3'UTR which recognize AUBPs and which are modulated by antisense transcripts.