The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

<p>Abstract</p> <p>Background</p> <p><it>CDK5R1 </it>plays a central role in neuronal migration and differentiation during central nervous system development. <it>CDK5R1 </it>has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of <it>CDK5R1 </it>3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of <it>CDK5R1 </it>expression.</p> <p>Results</p> <p>The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of <it>CDK5R1 </it>3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins.</p> <p>Conclusion</p> <p>Our findings evince the presence of both destabilizing and stabilizing regulatory elements in <it>CDK5R1 </it>3'-UTR and support the hypothesis that <it>CDK5R1 </it>gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of <it>CDK5R1 </it>expression. The fine tuning of <it>CDK5R1 </it>expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.</p

The 3' untranslated region of human contains regulatory elements affecting transcript stability-4

<p><b>Copyright information:</b></p><p>Taken from "The 3' untranslated region of human contains regulatory elements affecting transcript stability"</p><p>http://www.biomedcentral.com/1471-2199/8/111</p><p>BMC Molecular Biology 2007;8():111-111.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222623.</p><p></p> element (nt 2637–2687) was deleted (hatched bar) from the pGL4.71P-C6 in construct pGL4.71P-C6del by PCR. B) Luciferase activity of the two chimeric constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value). C) mRNA levels of the chimeric transcripts. Total RNA was extracted from the cells harvested 24 hours post-transfection, and the reporter gene mRNA levels were analyzed by RealTime PCR. mRNA levels of the chimeric reporter gene normalized as described, are represented as a percentage of the mRNA levels observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase mRNA values were obtained from at least three independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value)

The 3' untranslated region of human contains regulatory elements affecting transcript stability-3

<p><b>Copyright information:</b></p><p>Taken from "The 3' untranslated region of human contains regulatory elements affecting transcript stability"</p><p>http://www.biomedcentral.com/1471-2199/8/111</p><p>BMC Molecular Biology 2007;8():111-111.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222623.</p><p></p>nds). SK-N-BE cells were transiently transfected with reporter gene constructs and the pGL3 (firefly luciferase) vector. 24 hours after transfection (time = 0 h), cells were treated with DRB. Total RNA was extracted after various time-points. RNA was reverse-transcribed and RealTime RT-PCR was performed. Renilla luciferase transcript levels were normalized to GAPDH and firefly luciferase mRNAs. Data are expressed as percentage of RNA remaining after DRB addition and are representative of three independent experiments. B) Schematic representation of luciferase constructs carrying C2 fragment and sub-fragments. C) Luciferase activity of the chimeric constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value). D) mRNA levels of the chimeric transcripts. Total RNA was extracted from the cells harvested 24 hours post-transfection, and the reporter gene mRNA levels were analyzed by RealTime PCR. mRNA levels of the chimeric reporter gene normalized as described, are represented as a percentage of the mRNA levels observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase mRNA values were obtained from at least three independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value)

The 3' untranslated region of human contains regulatory elements affecting transcript stability-5

<p><b>Copyright information:</b></p><p>Taken from "The 3' untranslated region of human contains regulatory elements affecting transcript stability"</p><p>http://www.biomedcentral.com/1471-2199/8/111</p><p>BMC Molecular Biology 2007;8():111-111.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222623.</p><p></p>n neuronal ELAV and the irrelevant IgG antibodies. The bound mRNAs were analyzed by RT-PCR with primer pairs specific for (left panel) and GAP-43 (right panel). MW, Molecular weight marker; +, positive control (total SH-SY5Y cDNA); -, negative control (no sample). B) In vitro RNA-protein binding experiments were performed by UV cross-linking of different P-radiolabelled regions from 3'UTR and brain protein lysate. A selective immunoprecipitation of the formed mRNP complex was performed using the nELAV antibody (lanes 2–5). As a negative control a mix of all the different riboprobes was used and immunoprecipitated by the IgG antibody (-, lane 1), while GAP-43 3'UTR riboprobe was used as a positive control (+, lane 5)

The 3' untranslated region of human contains regulatory elements affecting transcript stability-0

<p><b>Copyright information:</b></p><p>Taken from "The 3' untranslated region of human contains regulatory elements affecting transcript stability"</p><p>http://www.biomedcentral.com/1471-2199/8/111</p><p>BMC Molecular Biology 2007;8():111-111.</p><p>Published online 3 Dec 2007</p><p>PMCID:PMC2222623.</p><p></p>ol. Regions of at least 100 bp with identity greater than 70% are indicated in pink. The plots refer to the human 3'-UTR sequence (UTRdb ID: 3HSA086450). The position of the predicted regulatory elements is indicated. B) Multiple alignment of the last portion of human (Hs.), mouse (Mm.), rat (Rn.) and zebrafish (Dr.) 3'-UTR. Stars represent fully conserved positions; the best ARE consensus is boxed in gray, the polyA sites are in bold. C) Representation of the most stable secondary structure of the human 3'-UTR as predicted by the Sfold algorithm. The magnification shows the loop (arrow) corresponding to the ARE with the best consensus (nt 2659–2671)