CDK5R1 encodes p35, an activator of CDK5, a proline-directed serine/threonine kinase that phosphorylates proteins involved in CNS development and maintenance. CDK5 and p35 were found to show an important role in neuronal migration and differentiation during CNS development and were also implicated in some neurodegenerative and cognitive disorders. Both the CDK5R1 3’-UTR remarkable size and its conservation during the evolution are strongly indicative of an important function in post-transcriptional regulation. To establish whether microRNAs have a role in CDK5R1 regulation, we carried out a prediction of miRNA target sites and, following expression analysis of specific miRNAs in comparison to CDK5R1/p35 expression, we selected for functional studies miR- 103 and miR-107. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection with the miR-103 or miR-107 precursor. Conversely, p35 levels significantly increased following transfection of the antagonists. Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 with an opposite shift following anti-miR-107 transfection, suggesting a direct action on translation efficiency. We finally identified, by luciferase assays, a specific target site where miR-103 and miR-107 are able to directly interact with the CDK5R1 3’-UTR. We recently reported that CDK5R1 3’-UTR contains regulatory elements affecting transcript stability. In particular, a 138 bp region, that doesn’t contain miRNA binding sites, has been identified as the most destabilizing portion of the 3’-UTR by luciferase assays. UV cross-linking and site directed mutagenesis experiments allowed us to delimit the binding site for RNA binding proteins (RBPs), among which the nELAV, showing a stabilizing activity on CDK5R1 transcript after overexpression and silencing experiments. To search for putative destabilizing factors, pull-down experiments have been carried out, allowing us to identify further binding factors, among which hnRNPA2/B1. The validation of hnRNPA2/B1 binding and the study of its silencing/overexpression might help to disclose the possible role of this protein on CDK5R1 post-transcriptional regulation.

IDENTIFICATION AND FUNCTIONALCHARACTERIZATION OF TRANS-ACTING FACTORSINVOLVED IN THE POST-TRANSCRIPTIONALREGULATION OF CDK5R1 / P. Zuccotti ; tutor: P. V. Riva ; coordinatore: M. Bolognesi. Universita' degli Studi di Milano, 2012 Jan 23. 24. ciclo, Anno Accademico 2011.

IDENTIFICATION AND FUNCTIONALCHARACTERIZATION OF TRANS-ACTING FACTORSINVOLVED IN THE POST-TRANSCRIPTIONALREGULATION OF CDK5R1

P. Zuccotti
2012

Abstract

CDK5R1 encodes p35, an activator of CDK5, a proline-directed serine/threonine kinase that phosphorylates proteins involved in CNS development and maintenance. CDK5 and p35 were found to show an important role in neuronal migration and differentiation during CNS development and were also implicated in some neurodegenerative and cognitive disorders. Both the CDK5R1 3’-UTR remarkable size and its conservation during the evolution are strongly indicative of an important function in post-transcriptional regulation. To establish whether microRNAs have a role in CDK5R1 regulation, we carried out a prediction of miRNA target sites and, following expression analysis of specific miRNAs in comparison to CDK5R1/p35 expression, we selected for functional studies miR- 103 and miR-107. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection with the miR-103 or miR-107 precursor. Conversely, p35 levels significantly increased following transfection of the antagonists. Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 with an opposite shift following anti-miR-107 transfection, suggesting a direct action on translation efficiency. We finally identified, by luciferase assays, a specific target site where miR-103 and miR-107 are able to directly interact with the CDK5R1 3’-UTR. We recently reported that CDK5R1 3’-UTR contains regulatory elements affecting transcript stability. In particular, a 138 bp region, that doesn’t contain miRNA binding sites, has been identified as the most destabilizing portion of the 3’-UTR by luciferase assays. UV cross-linking and site directed mutagenesis experiments allowed us to delimit the binding site for RNA binding proteins (RBPs), among which the nELAV, showing a stabilizing activity on CDK5R1 transcript after overexpression and silencing experiments. To search for putative destabilizing factors, pull-down experiments have been carried out, allowing us to identify further binding factors, among which hnRNPA2/B1. The validation of hnRNPA2/B1 binding and the study of its silencing/overexpression might help to disclose the possible role of this protein on CDK5R1 post-transcriptional regulation.
23-gen-2012
Settore BIO/13 - Biologia Applicata
CDK5R1 ; 3'UTR ; RBPs
RIVA, PAOLA VANDA
BOLOGNESI, MARTINO
Doctoral Thesis
IDENTIFICATION AND FUNCTIONALCHARACTERIZATION OF TRANS-ACTING FACTORSINVOLVED IN THE POST-TRANSCRIPTIONALREGULATION OF CDK5R1 / P. Zuccotti ; tutor: P. V. Riva ; coordinatore: M. Bolognesi. Universita' degli Studi di Milano, 2012 Jan 23. 24. ciclo, Anno Accademico 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/168394
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