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Post-transcriptional regulation mediated by ELAV/Hu RNA-binding proteins in motor neuron-like NSC34 cells
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Post-trascriptional regulation of neuro-oncological ventral antigen 1 by the neuronal RNA-binding proteins ELAV
No full textAlternative splicing of pre-mRNAs plays an important role in generating biological and functional diversity. Neuro-oncological ventral antigen 1 (Nova1) is a neuron-specific splicing factor that controls the alternative processing of a wide array of mRNAs important for synaptic activity. It is essential for the proper development of the mammalian motor system and for the survival of motoneurons. Because Nova1 gene contains putative regulatory AU-rich elements (ARE) in its highly conserved 3''-untranslated region, we investigated whether its expression is regulated by post-transcriptional mechanisms mediated by ARE-binding proteins. Among these, the neuronal ELAV (nELAV) factors are interesting candidates, because their RNA binding activity is necessary for neuronal differentiation and maintenance. By analysis of ribonucleoprotein complexes in vivo and in vitro we demonstrated that the Nova1 mRNA is a novel target of the nELAV proteins. We defined the nELAV binding site by functional experiments with luciferase reporter gene and Nova1 3''-untranslated region deletion sequences. Gene silencing and overexpression of the nELAV member HuD in motoneuronal NSC34 cells indicate that Nova1 mRNA stability and translation are positively and strongly controlled by the nELAV proteins. In addition, nELAV phosphorylation by a PKC-dependent pathway induces the recruitment of Nova1 mRNA to polysomes. Noteworthy, we found that nELAV proteins are also able to modulate Nova1 splicing activity on its target genes. Our data indicate nELAV proteins as the first factors affecting the expression and activity of the neuronal splicing regulator Nova1 and, consequently, as major candidates for the physiological modulation of Nova1-dependent processing of pre-mRNAs in neurons
Post-transcriptional regulation of neuro-oncological ventral antigen 1 by the neuronal RNA-binding proteins ELAV
No full textAlternative splicing of pre-mRNAs plays an important role in
generating biological and functional diversity. Neuro-oncological
ventral antigen 1 (Nova1) is a neuron-specific splicing factor
that controls the alternative processing of a wide array of
mRNAs important for synaptic activity. It is essential for the
proper development of the mammalian motor system and for
the survival of motoneurons. Because Nova1 gene contains
putative regulatory AU-rich elements (ARE) in its highly conserved
3-untranslated region, we investigated whether its
expression is regulated by post-transcriptional mechanisms
mediated by ARE-binding proteins. Among these, the neuronal
ELAV (nELAV) factors are interesting candidates, because their
RNA binding activity is necessary for neuronal differentiation
and maintenance. By analysis of ribonucleoprotein complexes
in vivo and in vitro we demonstrated that the Nova1 mRNA is a
novel target of the nELAV proteins. We defined the nELAV
binding site by functional experiments with luciferase reporter
gene and Nova1 3-untranslated region deletion sequences.
Gene silencing and overexpression of the nELAV member HuD
in motoneuronal NSC34 cells indicate that Nova1mRNAstability
and translation are positively and strongly controlled by the
nELAVproteins. In addition,nELAVphosphorylation by aPKCdependent
pathway induces the recruitment of Nova1mRNAto
polysomes. Noteworthy, we found that nELAV proteins are also
able to modulate Nova1 splicing activity on its target genes. Our
data indicate nELAV proteins as the first factors affecting the
expression and activity of the neuronal splicing regulator Nova1
and, consequently, as major candidates for the physiological
modulation of Nova1-dependent processing of pre-mRNAs in
neurons
TDP-43 is recruited to stress granules in conditions of oxidative insult
No full textTAR DNA-binding protein 43 (TDP-43) forms abnormal ubiquitinated and phosphorylated inclusions in brain tissues from patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. TDP-43 is a DNA/RNA-binding protein involved in RNA processing, such as transcription, pre-mRNA splicing, mRNA stabilization and transport to dendrites. We found that in response to oxidative stress and to environmental insults of different types TDP-43 is capable to assemble into stress granules (SGs), ribonucleoprotein complexes where protein synthesis is temporarily arrested. We demonstrated that a specific aminoacidic interval (216-315) in the C-terminal region and the RNA-recognition motif 1 domain are both implicated in TDP-43 participation in SGs since their deletion prevented the recruitment of TDP-43 into SGs. Our data show that TDP-43 is a specific component of SGs and not of processing bodies, although we proved that TDP-43 is not necessary for SG formation, and its gene silencing does not impair cell survival during stress. The analysis of spinal cord tissue from ALS patients showed that SG markers are not entrapped in TDP-43 pathological inclusions. Although SGs were not evident in ALS brains, we speculate that an altered control of mRNA translation in stressful conditions may trigger motor neuron degeneration at early stages of the disease
The 3' untranslated region of human contains regulatory elements affecting transcript stability
No full textBACKGROUND: CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. RESULTS: The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 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. CONCLUSION: Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and 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
Neural-specific RNA-binding proteins and cell fate specification in neural progenitors : an "in vitro" and "in vivo" analysis
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