title: hnRNPA2/B1 and nELAV proteins bind to a specific U-rich element in CDK5R1 3'-UTR and oppositely regulate its expression.
abstract: Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of cyclin-dependent kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138 bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73 bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not vice versa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression.
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Zuccotti
authors.affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy; IRCCS Istituto Auxologico Italiano, Cusano, Milan, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Colombrita
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Moncini
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Barbieri
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Lunghi
authors.affiliation: Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy; IBFM-CNR, Segrate, Milan, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gelfi
authors.affiliation: Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy; IBFM-CNR, Segrate, Milan, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: De Palma
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Nicolin
authors.affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy; IRCCS Istituto Auxologico Italiano, Cusano, Milan, Italy.
authors.forename: Antonia
authors.initials: A
authors.lastname: Ratti
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy. Electronic address: marco.venturin@unimi.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Venturin
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy. Electronic address: paola.riva@unimi.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Riva
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D015153:Blotting, Western; D002454:Cell Differentiation; D051959:ELAV Proteins / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
chemical_list: D020413:3' Untranslated Regions; D051959:ELAV Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D038621:Regulatory Sequences, Ribonucleic Acid; C089495:neuronal Cdk5 activator (p25-p35); D008156:Luciferases
keywords: CDK5R1/p35; Post-transcriptional regulation; RNA binding protein; U-rich element; hnRNPA2/B1; nELAV