Functional study of regulatory elements in CDK5R1 3’-UTR: evidence on post-transcriptional gene expression regulation
Tesi di Dottorato
Data di Pubblicazione:
2007
Citazione:
Functional study of regulatory elements in CDK5R1 3’-UTR: evidence on post-transcriptional gene expression regulation / S. Moncini ; Tutor: P. V. Riva; coordinatore: G. Zanetti. ex-DIPARTIMENTO DI BIOLOGIA E GENETICA PER LE SCIENZE MEDICHE, ex-DIPARTIMENTO DI FARMACOLOGIA, CHEMIOTERAPIA E TOSSICOLOGIA MEDICA, 2007 Dec 19. 20. ciclo, Anno Accademico 2006/2007.
Abstract:
Background
CDK5R1 encodes for p35, a neuron-specific activator of cyclin-dependent kinase 5 (CDK5), whose activity plays a central role in neuronal migration during central nervous system development. Cdk5r KO mice have severe cortical lamination defects and suffer from adult mortality and seizures. The active CDK5-p35 complex is involved in several processes required for central nervous system development and functioning, as axonal regeneration, cellular differentiation, neuronal apoptosis, learning and memory processes, synaptic transmission and membrane trafficking during the outgrowth of neuronal processes. Moreover, increased CDK5 activation by p25, a proteolytic fragment containing the C-terminal portion of p35, has been implicated in the pathogenesis of several neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. CDK5R1 has been proposed as a candidate gene for mental retardation susceptibility in NF1 microdeletion syndrome.
CDK5R1 spans for 4.17 kb on chr 17q11.2, and its coding region (1021 bp) consists of a single exon. In addition, the CDK5R1 gene displays a large 3’-untranslated region (3’-UTR). The remarkable size of CDK5R1 3’-UTR suggests a role in post-transcriptional regulation of CDK5R1 expression. Untranslated regions (UTRs) are known to play crucial roles in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNA out of the nucleus, and of the translation efficiency, subcellular localization and stability. The importance of 3’-UTRs in regulating gene expression is underlined by the finding that mutations which alter the 3’-UTR can lead to serious pathology. Nucleotide patterns or motifs located in 3' UTRs can interact with specific RNA-binding proteins. The biological activity of regulatory motifs at the RNA level relies on a combination of primary and secondary structure. Interactions between sequence elements located in the 3’-UTRs and specific microRNAs have also been shown to play key regulatory roles.
Results
The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs) and a GY-box element. The GY−box (GTCTTCC) motif, described in many 3'−UTRs of genes involved in Notch signalling in Drosophila is likely to be involved in the formation of RNA duplexes with complementary sequences at the 5' ends of some Drosophila microRNAs in vivo. AU-rich sequences, function as potent destabilizing elements that cause rapid decay of the respective transcript; these elements are composed of a variable number of copies of the AUUUA pentamer or UUAUUUAUU nonamer. Among the predicted AREs in CDK5R1, the nt 2659-2671 ARE shows complete identity to the consensus sequence for Class I AREs, according to the ARED 3.0 definition, is highly conserved in mammals and zebrafish and is predicted to be accessibile to the binding of trans-acting factors.
The effect of the 3’-UTR on gene expression was studied with the Dual-Luciferase reporter assay. The insertion of CDK5R1 3’-UTR into luciferase 3’-UTR caused a decreased luciferase activity and mRNA level in four transfected cell lines (SK-N-BE, SH-SY5Y, HEK-293 and MCF-7). The dissection of 3’-UTR into 6 fragments (C1-6), each containing at least one predicted regulatory element, allowed us to investigate the potential role of each region. All the chimeric constructs showed, in most of the studied cell lines, a general decrease of luciferase activity. In most cases these effects are likely to involve transcript stability rather than translational repression mechanisms, since reduced reporter activity levels corresponded to reduced mRNA levels. A region (C2), leading to a very strong mRNA destabiliza
Tipologia IRIS:
13 - Tesi di dottorato discussa entro ottobre 2010
Elenco autori:
S. Moncini
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