Functional study of transcription cis-regulatory elements predicted in the CDK5R1 3’UTR

CDK5R1 encodes for p35, a neuron-specific activator of cyclindependent kinase 5 (CDK5), whose activity plays a central role in neuronal migration during CNS development and which has been implicated in several neurodegenerative disorders. The remarkable size of CDK5R1 3’UTR prompted us to search for UTR regulatory elements which act on mRNA stability and translational efficiency, by means of the UTRScan bioinformatic tool. We predicted eight possible ARE (AU-Rich Elements), involved in transcript deadenylation/degradation, and a GY-box element, known to have a role in Drosophila post-transcriptional negative regulation of gene expression. A Dual Luciferase assay was used to carry out the functional analysis: we cotransfected in SK-N-BE and HEK-293 cellular lines a Firefly luciferase expressing control plasmid and six overlapping fragments, covering the entire CDK5R1 3’UTR (C1-6), cloned in plasmids expressing Renilla reniformis luciferase at the 3’ end of the reporter gene. ARE containing C1 and C2 fragments showed a decreased luciferase activity in both cell lines, while ARE containing C5 and C6 fragments displayed similar levels of luciferase activity, compared to the control plasmid. The C3 fragment, covering the GY-box, showed high luciferase activity, suggesting for this element a function of transcript stabilizer in human cells, differently from that evidenced in Drosophila. The ARE fragment C4, displayed reduced luciferase levels only in SK-N-BE cells, suggesting a line-specific post-translational regulation control. The contribution of the predicted regulatory elements on posttranscriptional regulation mechanisms will be further elucidated by studying deleted/mutated fragments and performing degradation assays.