Identification of a potential regulatory element forming a hairpin structure within the 3’UTR of CDK5R1

CDK5R1 encodes for p35, a regulatory subunit of CDK5 kinase, which is fundamental for normal neural development and function. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3’UTR, which is highly conserved in mammals and contains AREs and miRNA target sites, suggests a role in post-transcriptional regulation of its expression. The insertion of CDK5R1 3’UTR into luciferase gene causes a decreased luciferase activity in four transfected cell lines. A 3’UTR region (named C2) leads to a very strong luciferase mRNA reduction, owing to a significantly lower half-life, indicating accelerated mRNA degradation. This fragment was dissected into smaller regions and a 65 bp sequence (C2.11), in which no known regulatory elements were predicted, has been identified to be responsible of the decreased gene expression. A stable structural motif (forming a hairpin) of C2.11 fragment was predicted by RnaProfile and SFold programs, both starting from the isolated fragment and considering it within the whole 3’UTR. Lowering of luciferase levels for the construct with an intact hairpin structure in contrast with unchanged levels for four mutated/deleted structures suggests that this putative element might really have a regulatory role. Since complementary mutations restoring the hairpin did not affect luciferase activity, we suggest that both the sequence and the structure are essential for the ability of C2.11 fragment to reduce transcript stability. Further mutagenesis experiments, binding assays and RNAse protection assays will disclose the function of this novel CDK5R1 3’UTR regulatory element.