Identification of post-transcriptional regulatory elements in CDK5R1 3’UTR gene involved in CNS development and functioning

CDK5R1 encodes p35, an activator of CDK5, which is involved in neuronal migration and differentiation during CNS development. It has been implicated in some neurodegenerative disorders and candidate for the non-specific mental retardation. The considerable size of CDK5R1 3’UTR and the high level of sequence conservation suggests a role in post-transcriptional regulation. We recently reported that the large CDK5R1 3’UTR contains some regulatory elements affecting transcript stability. Precisely, a 138 bp region was shown to be the most destabilizing region within the 3’UTR. We search for posttranscriptional destabilizing factors in this region by UV-cross-linking that showed the binding of some RBPs among which the strongest band had a molecular weight compatible with that of the nELAV confirmed by immunoprecipitation. The fragment has been further divided in four regions for UV-crosslinking experiment that allowed to identify a stretch of 7 bp binding region and through the site-directed mutagenesis we identified the binding sequence. Silencing and overexpression of this factor are in progress in order to validate its destabilizing activity on CDK5R1 expression. To verify the binding of further regulatory elements the four regions of the 138 bp fragment have been transcribed and used for pulldown experiment of protein extracts from SK-N-BE cells. Following mass spectrometry analysis we identified the hnRNPA2/B1 for which the cytoplasmatic function is unknown and should be confirmed by immunoprecipitation assays. This study, besides defining the regulatory mechanisms of CDK5R1 expression, may help to identifying the pathogenetic implications of the gene in neurodegenerative and cognitive diseases