1,720,978 research outputs found
Functional study of regulatory elements in CDK5R1 3’-UTR: evidence on post-transcriptional gene expression regulation
No full textBackground
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 destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. This fragment was dissected into smaller regions and a 65 bp (named C2.11) sequence has been identified to be responsable of the decreased gene expression of the all C2 fragment, in which none regulatory elements were predicted. The existence of a stable structural motif (forming a hairpin) was predicted by both RnaProfile and SFold programs in both the analysed entire 3'-UTR and C2.11 transcripts, and it was speculated that it may have a regulatory role. We show here that the hairpin structure within the 3'-UTR influences the expression of the luciferase reporter gene by means of generation of mutants. Lowering of luciferase levels for the construct with an intact hairpin structure in contrast with almost unchanged levels for the four mutated/deleted structures confirm the
importance of this sequence and suggest that its disruption may directly affect rapid mRNA degradation. Since complementary mutations restoring the hairpin structure did not restore luciferase activity, we suggest that sequence within a structure is essential for the ability of the C2.11 fragment to reduce luciferase activity.
The generation of a construct with the deletion of the canonical GY-box motif revealed the inactivity of this element in all the cell lines used for the transfection experiments.
The 3’ end of the transcript (C6 fragment), containing the class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. The deletion of the canonical nt 2659-2671 ARE in the C6 fragment reduced luciferase activity mRNA levels in all the analyzed cell lines, including SK-N-BE and SH-SY5Y, strongly suggesting a stabilizing role of the canonical element in neuroblastoma-derived cells through the binding of neuronal-specific stabilizing factors.
We also observed the interaction of the stabilizing neuronal RNA-binding proteins nELAV with the CDK5R1 transcript in SH-SY5Y cells by immunoprecipitation and UV cross-linking experiments allowed us to observe that C1, C2 and C6 subregions show affinity for nELAV proteins in vitro.
microRNA (miRNA) target site prediction with PicTar software found several target sites along the entire CDK5R1 3’-UTR without clustering. Between the 20 miRNAs predicted to bind CDK5R1, miR-15a, miR-103 and miR-107 present a high number of target sites with a free energy <-20 kcal/mol which point to these three miRNAs as the most probable to be functional. Nine pre-miRNAs have been shown to be expressed in the cell lines of interest. A preliminary quantitative analysis points to an inverse correlation of expression between miR-107 and the proteic product of CDK5R1, p35, that correlates with the negative role of miRNA on expression of proteins encoded by their target transcripts.
Conclusions
Our findings have shown the presence of several regulatory elements in CDK5R1 3’-UTR, and for a few of them we found a destabilizing or stabilizing function. The 3’-UTR seems to contain some regulatory elements implicated in rapid mRNA turnover which, as a consequence, maintain the steady-state transcript at low levels, and others which have a cell-specific stabilizing effect on the transcript that may contribute to rapidly increase the expression of CDK5R1 during specific biological processes. Our findings also support the
hypothesis that CDK5R1 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 CDK5R1 expression. The fine tuning of CDK5R1 expression by 3’-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders. The large 3’-UTR of CDK5R1 is expected to contain further cis-regulatory elements and interact with further trans-acting molecules, creating the possibility of complex gene expression modulation
Identification of a potential regulatory element forming a hairpin structure within the 3’UTR of CDK5R1
No full textCDK5R1 encodes for p35, a regulatory subunit of CDK5 kinase, which is fundamental for normal neural development and function. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3’UTR, which is highly conserved in mammals and contains AREs and miRNA target sites, suggests a role in post-transcriptional regulation of its expression. The insertion of CDK5R1 3’UTR into luciferase gene causes a decreased luciferase activity in four transfected
cell lines. A 3’UTR region (named C2) leads to a very strong luciferase mRNA reduction, owing to a significantly lower half-life, indicating accelerated mRNA degradation. This fragment was dissected into smaller
regions and a 65 bp sequence (C2.11), in which no known regulatory elements were predicted, has been identified to be responsible of the decreased gene expression. A stable structural motif (forming a hairpin) of C2.11 fragment was predicted by RnaProfile and SFold programs, both starting from the isolated fragment and considering it within the whole 3’UTR. Lowering of luciferase levels for the construct with an intact hairpin structure in contrast with unchanged levels for
four mutated/deleted structures suggests that this putative element might really have a regulatory role. Since complementary mutations restoring the hairpin did not affect luciferase activity, we suggest that
both the sequence and the structure are essential for the ability of C2.11 fragment to reduce transcript stability. Further mutagenesis experiments, binding assays and RNAse protection assays will disclose the function of this novel CDK5R1 3’UTR regulatory element
Functional study of transcription cis-regulatory elements predicted in the CDK5R1 3’UTR
No full textCDK5R1 encodes for p35, a neuron-specific activator of cyclin-dependent 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 post-transcriptional regulation mechanisms will be further elucidated by studying deleted/mutated fragments and performing degradation assays
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome
Full text linkBackground Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish.
Methods and results The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients.
Conclusions Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects
Mutations and novel polymorphisms in coding regions and UTRs of CDK5R1 and OMG genes in patients with nonsyndromic mental retardation
Full text linkMental retardation (MR) is displayed by 57% of
NF1 patients with microdeletion syndrome as a result of
17q11.2 region haploinsufficiency. We considered the
cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1)
and oligodendrocyte-myelin glycoprotein (OMG) genes,
mapping in the NF1 microdeleted region, as candidate
genes for MR susceptibility. CDK5R1 encodes for a
neurone-specific activator of cyclin-dependent kinase 5
(CDK5) involved in neuronal migration during central
nervous system development. OMG encodes for an
inhibitor of neurite outgrowth by the binding to the
Nogo-66 receptor (RTN4R). CDK5R1 and OMG genes
are characterized by large 3′ and 5′ untranslated regions
(UTRs), where we predict the presence of several transcription/
translation regulatory elements. We screened
100 unrelated Italian patients affected by unspecific MR
for mutations in CDK5R1 and OMG coding regions and in
their 3′ or 5′ UTRs. Four novel mutations and two novel
polymorphisms for CDK5R1 and three novel mutations
for OMG were detected, including two missense changes
(c.323C>T; A108V in CDK5R1 and c.1222A>G; T408A
in OMG), one synonymous codon variant (c.532C>T;
L178L in CDK5R1), four variants in CDK5R1 3′UTR and
two changes in OMG 5′UTR. All the mutations were absent in 370 chromosomes from normal subjects. The
allelic frequencies of the two novel polymorphisms in
CDK5R1 3′UTR were established in both 185 normal and
100 mentally retarded subjects. Prediction of mRNA and
protein secondary structures revealed that two changes
lead to putative structural alterations in the mutated
c.2254C>G CDK5R1 3′UTR and in OMG T408A gene
product
The human CDK5R1 3'UTR contains distinct subregions affecting transcript stability
No full textHuman CDK5R1 encodes for p35, a neurone-specific activator of
CDK5, which is involved in neuronal migration and differentiation
during CNS development. CDK5R1 has been implicated in neurodegenerative
disorders and proposed as a candidate gene for mental
retardation. The remarkable size of CDK5R1 3’UTR suggests a role
of this region in the control of CDK5R1 expression by post-transcriptional
regulatory elements modulating mRNA stability or translation efficiency.
Bioinformatic analysis showed a high conservation degree in
mammals and predicted several AU-Rich Elements (AREs). CDK5R1
3’UTR was cloned in pGL4.71 at the 3’ end of the Renilla luciferase reporter
gene to perform Dual Luciferase assays: the construct showed
a decreased luciferase activity in six transfected cell lines. The quantitative
analysis of luciferase mRNA suggests that CDK5R1 3’UTR affects
mRNA stability. We identified five 3’UTR subregions reducing the
luciferase activity in some instance with a cell line-dependent way. A
region showed a significantly low halflife, suggesting an accelerated
mRNA degradation. We also identified, by deletion analysis, a type I
ARE displaying a stabilizing effect in two neuroblastoma cell lines. Our
findings evince the presence of both destabilizing and stabilizing regulatory
elements in CDK5R1 3’UTR. We are now attempting to identify,
by REMSA and immunoprecipitation assays, stabilizing neuronal proteins
that specifically bind the type I ARE, with the final aim of verifying
the functionality of this element. The fine tuning of CDK5R1 expression
by 3’UTR may play a role in CNS development and functioning, with
potential implications in neurodegenerative and cognitive disorders
Differential allelic expression of the SOS1 c.755C activating variant in a Noonan syndrome family
No full textNoonan Syndrome (NS) is a genetic condition characterized by congenital heart defects, short stature and
characteristic facial features. We analyzed a girl with moderate learning disabilities, delayed language
development, craniofacial features and skin anomalies reminiscent of NS. After a mutation screening of the
known NS genes PTPN11, SOS1, RAF1, KRAS, GRB2, BRAF and SHOC2 we found the heterozygous
c.755T/C variation in SOS1 causing the I252T substitution, which was considered possibly pathogenetic by
bioinformatic predictions. The same mutation was present in the proband’s mother and maternal grandfather,
both displaying some NS features, but also by a healthy subject on 1000 genomes analyzed. The functional
analysis revealed that the SOS1 c.755T/C activated the Ras effector Erk1, confirming the predicted
pathogenetic substitution. To explain the incomplete penetrance of the reported mutation we hypothesized
that SOS1 may be subjected to a differential allelic expression (DAE). Interestingly, after sequencing the
cDNA from peripheral blood compared to genomic DNA, we showed a DAE of some known SOS1 SNSs in
healthy individuals and observed the mutated allele C 50% more expressed than the normal allele T in all our
NS familial carriers. The similar level of SOS1 mRNA, between mutated and control individuals, suggests that
the mutation here described does not affect SOS1 expression. We are now evaluating the SOS1 promoter
polymorphisms. This study, providing the first evidence of allelic imbalance of SOS1, pinpoint DAE as a
possible mechanism underlying a different penetrance of some SOS1 mutated alleles in unrelated carriers
- …
