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Cited by 112RNA Research and SplicingRNA regulation and diseaseDevelopmental Biology and Gene Regulation

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Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

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Mansfield, Kyle D., Keene, Jack D.·2012·Nucleic Acids Research
DOI 10.1093/nar/gkr1114PMC PMC3315332
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Abstract

The ubiquitously expressed RNA-binding protein HuR increases the stability and translation of mRNAs encoding growth regulatory proteins that promote proliferation in a variety of cell types. However, the three neuron-specific ELAV/Hu proteins, HuB, HuC and HuD, while binding to the same types of mRNAs, are required instead for neuronal differentiation, and it becomes difficult to reconcile these contrary functions when all four Hu proteins are expressed in the same neuron. HuR mRNA exists as three alternatively polyadenylated variants, a 1.5-kb testes-specific mRNA isoform, a ubiquitous 2.4-kb isoform and a 6.0-kb isoform that we now show is induced during neuronal differentiation and appears to be neuron-specific. This 6.0-kb neuron-specific mRNA isoform is inherently less stable and produces less HuR protein than the ubiquitous 2.4-kb mRNA. Furthermore, we show that neuronal HuB, HuC and HuD, as well as HuR itself, can bind at the 2.4-kb mRNA polyadenylation site, and when overexpressed can affect alternative polyadenylation to generate an extended HuR 3'-UTR that is translationally suppressed. We propose that the regulation of HuR protein expression by alternative polyadenylation allows neurons to post-transcriptionally regulate mRNAs-encoding factors required for proliferation versus differentiation to facilitate neuronal differentiation.

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Figures

Figure 1. Diverse mRNA isoforms of HuR are expressed in specific cell types. (A and B) Probe sets from the GeneAtlas MOE430 (gcrma) data set that exclusively recognize the HuR 6.0-kb mRNA isoform (A, probe 1452858_at) or ‘pan-HuR’ mRNA (B, probe 1448151_at) were analyzed for relative expression levels across murine tissues grouped according to bodily system (Digest = Digestive, Repro = Reproductive; identity of tissues can be found in Supplementary Table S1). (C) Tissue expression data for probes corresponding to the various Hu family members was downloaded from the BioGPS gene portal hub (http://www.biogps.gnf.org) and analyzed in JMP. Shown is the upper triangular matrix of Spearman correlation coefficients for all pair-wise comparisons of expression values of probes specific for the various Hu family members including the 6.0-kb mRNA isoform of HuR.
Figure 1. Diverse mRNA isoforms of HuR are expressed in specific cell types. (A and B) Probe sets from the GeneAtlas MOE430 (gcrma) data set that exclusively recognize the HuR 6.0-kb mRNA isoform (A, probe 1452858_at) or ‘pan-HuR’ mRNA (B, probe 1448151_at) were analyzed for relative expression levels across murine tissues grouped according to bodily system (Digest = Digestive, Repro = Reproductive; identity of tissues can be found in Supplementary Table S1). (C) Tissue expression data for probes corresponding to the various Hu family members was downloaded from the BioGPS gene portal hub (http://www.biogps.gnf.org) and analyzed in JMP. Shown is the upper triangular matrix of Spearman correlation coefficients for all pair-wise comparisons of expression values of probes specific for the various Hu family members including the 6.0-kb mRNA isoform of HuR.CC BY-NC · PubMed Central
Figure 2. Expression analysis of neuronal ELAV/Hu family members during retinoic acid induced differentiation of mouse P19 embryonic carcinoma cells. (A) Phase contrast micrograph of P19 cells (P19) treated with 5 µM Retinoic Acid (RA) for 4 days before being replated and grown for an additional 2 days (4/2) showing the change in morphology and formation of neuronal processes after RA treatment. (B and C) Real time PCR bar graphs validating the induction of neuronal differentiation via increased expression of neuronal markers (HuB, HuC, HuD, NGC), the glial marker GFAP (B) and decreased expression of the stem cell marker Oct3 (C). GapDH mRNA level was used as a control. Values were calculated as fold change ratios using ΔΔCt values ±SD. The means and SD's are represented from three independent experiments.
Figure 2. Expression analysis of neuronal ELAV/Hu family members during retinoic acid induced differentiation of mouse P19 embryonic carcinoma cells. (A) Phase contrast micrograph of P19 cells (P19) treated with 5 µM Retinoic Acid (RA) for 4 days before being replated and grown for an additional 2 days (4/2) showing the change in morphology and formation of neuronal processes after RA treatment. (B and C) Real time PCR bar graphs validating the induction of neuronal differentiation via increased expression of neuronal markers (HuB, HuC, HuD, NGC), the glial marker GFAP (B) and decreased expression of the stem cell marker Oct3 (C). GapDH mRNA level was used as a control. Values were calculated as fold change ratios using ΔΔCt values ±SD. The means and SD's are represented from three independent experiments.CC BY-NC · PubMed Central
Figure 3. The 6.0-kb HuR mRNA isoform is expressed during retinoic acid induced neuronal differentiation of P19 embryonic carcinoma cells. P19 cells were differentiated in the presence (RA) or absence (Ctrl) of 5 µM RA for 4 days, then replated and allowed to differentiate for 2–6 days. At each time point, total RNA was isolated via Trizol. (A) Northern Blot showing increasing expression of the 6.0-kb mRNA isoform with either a neuron-specific probe (HuR NS from B) or a probe detecting all HuR mRNA isoforms (HuR ORF from B). β-Actin and ribosomal RNA (18S/28S) were used as loading controls. MW = molecular weight as determined by position of rRNA 18S and 28S bands (B) Semiquantitative PCR to confirm results of Northern blots shown in A. PCR products show consistent expression of the HuR ORF but increased expression of the 6.0-kb HuR neuron-specific mRNA isoform (HuR NS). Neuron-specific mRNAs HuC and Neuroligin 3 (Neuro3) were used to verify the progression of P19 neuronal differentiation. (C) Western blot analysis of Hu protein expression showing induction of neuronal-specific HuB and D during P19 differentiation. GapDH served as a loading control.
Figure 3. The 6.0-kb HuR mRNA isoform is expressed during retinoic acid induced neuronal differentiation of P19 embryonic carcinoma cells. P19 cells were differentiated in the presence (RA) or absence (Ctrl) of 5 µM RA for 4 days, then replated and allowed to differentiate for 2–6 days. At each time point, total RNA was isolated via Trizol. (A) Northern Blot showing increasing expression of the 6.0-kb mRNA isoform with either a neuron-specific probe (HuR NS from B) or a probe detecting all HuR mRNA isoforms (HuR ORF from B). β-Actin and ribosomal RNA (18S/28S) were used as loading controls. MW = molecular weight as determined by position of rRNA 18S and 28S bands (B) Semiquantitative PCR to confirm results of Northern blots shown in A. PCR products show consistent expression of the HuR ORF but increased expression of the 6.0-kb HuR neuron-specific mRNA isoform (HuR NS). Neuron-specific mRNAs HuC and Neuroligin 3 (Neuro3) were used to verify the progression of P19 neuronal differentiation. (C) Western blot analysis of Hu protein expression showing induction of neuronal-specific HuB and D during P19 differentiation. GapDH served as a loading control.CC BY-NC · PubMed Central
Figure 4. Effect of alternative polyadenylation on stability and translation of HuR mRNA. (A) The 6.0-kb neuron-specific (NS) HuR mRNA has a shorter half-life than the 2.4-kb ubiquitously expressed mRNA. Control (ctrl) or differentiated (RA) P19 cells were treated with the transcriptional inhibitor Actinomycin D and total RNA collected at the indicated times. HuR mRNA levels were determined via real time PCR using ΔΔCt calculations and the results plotted as percentage of the initial transcript remaining. Mean half lives were calculated via linear regression and shown ±SD from three independent experiments. (B) Representative immunoblot from HEK293T cells transiently transfected with equimolar amounts of constructs consisting of the HuR ORF fused to the indicated portion of the HuR 3′-UTR, showing decreased protein production from the neuron-specific 6.0-kb HuR mRNA isoform as compared to the ubiquitous 2.4-kb form. Topoisomerase (Topo) is shown as a loading control. (C) Western blot showing a progressive decrease in endogenous HuR protein expression in terminally differentiated P19 cells following treatment with the mitotic inhibitor AFC to eliminate non-neuronal cells. Semiquantitative PCR was used to determine relative expression of the HuR 6.0-kb mRNA isoform. (D) Western blot showing decreased endogenous HuR protein expression in isolated neuronal but not glial cells following P19 differentiation. Tubulin is shown as a loading control.
Figure 4. Effect of alternative polyadenylation on stability and translation of HuR mRNA. (A) The 6.0-kb neuron-specific (NS) HuR mRNA has a shorter half-life than the 2.4-kb ubiquitously expressed mRNA. Control (ctrl) or differentiated (RA) P19 cells were treated with the transcriptional inhibitor Actinomycin D and total RNA collected at the indicated times. HuR mRNA levels were determined via real time PCR using ΔΔCt calculations and the results plotted as percentage of the initial transcript remaining. Mean half lives were calculated via linear regression and shown ±SD from three independent experiments. (B) Representative immunoblot from HEK293T cells transiently transfected with equimolar amounts of constructs consisting of the HuR ORF fused to the indicated portion of the HuR 3′-UTR, showing decreased protein production from the neuron-specific 6.0-kb HuR mRNA isoform as compared to the ubiquitous 2.4-kb form. Topoisomerase (Topo) is shown as a loading control. (C) Western blot showing a progressive decrease in endogenous HuR protein expression in terminally differentiated P19 cells following treatment with the mitotic inhibitor AFC to eliminate non-neuronal cells. Semiquantitative PCR was used to determine relative expression of the HuR 6.0-kb mRNA isoform. (D) Western blot showing decreased endogenous HuR protein expression in isolated neuronal but not glial cells following P19 differentiation. Tubulin is shown as a loading control.CC BY-NC · PubMed Central
Figure 5. Label transfer experiments to determine sites of binding of Hu family members at the PolyA site of HuR mRNA. (A) Depiction of HuR mRNA sequence surrounding the 2.4-kb mRNA isoform polyadenylation signal and cleavage site (bolded and underlined). (B) Radiolabeled 50-bp RNA fragments (1–7 corresponding to sequence in A and depicted in D) were incubated with HEK293T extracts overexpressing the indicated constructs, UV cross-linked, immunoprecipitated and separated on SDS–PAGE gel to determine binding sites. (C) Specificity of binding was verified via cold competition with either a negative (Fragment 1) or positive (Fragment 4) probe. Band intensities from PhosphorImager were quantified and normalized for percent of control binding. (D) UCSC Genome Browser view of hELAVL1 (HuR) 3′-UTR showing published HuR binding sites near the 2.4-kb polyadenylation signal as determined by CLIP or PAR-CLIP analysis. Gel shift fragments used in this study are shown at top and the HuR 2.4-kb mRNA PolyA (PAS) and cleavage site (CS) are shown above the HuR 6.0-kb neural-specific (NS) and 2.4-kb (ubiq) mRNA for reference.
Figure 5. Label transfer experiments to determine sites of binding of Hu family members at the PolyA site of HuR mRNA. (A) Depiction of HuR mRNA sequence surrounding the 2.4-kb mRNA isoform polyadenylation signal and cleavage site (bolded and underlined). (B) Radiolabeled 50-bp RNA fragments (1–7 corresponding to sequence in A and depicted in D) were incubated with HEK293T extracts overexpressing the indicated constructs, UV cross-linked, immunoprecipitated and separated on SDS–PAGE gel to determine binding sites. (C) Specificity of binding was verified via cold competition with either a negative (Fragment 1) or positive (Fragment 4) probe. Band intensities from PhosphorImager were quantified and normalized for percent of control binding. (D) UCSC Genome Browser view of hELAVL1 (HuR) 3′-UTR showing published HuR binding sites near the 2.4-kb polyadenylation signal as determined by CLIP or PAR-CLIP analysis. Gel shift fragments used in this study are shown at top and the HuR 2.4-kb mRNA PolyA (PAS) and cleavage site (CS) are shown above the HuR 6.0-kb neural-specific (NS) and 2.4-kb (ubiq) mRNA for reference.CC BY-NC · PubMed Central
Figure 6. Overexpression of the neuron-specific Hu Family members (HuB, HuC and HuD) affects alternative polyadenylation of HuR mRNA. (A) Real time PCR results of transiently transfected HEK293T cells expressing the indicated FLAG tagged constructs demonstrating increased expression of alternatively polyadenylated HuR 6.0-kb mRNA, while maintaining overall HuR levels (Total). GapDH mRNA level was used as a control. Values were calculated as fold change ratios using ΔΔCT values ±SD. The means and SD's are represented from three independent experiments. Asterisks indicates P < 0.05 as determined by Fisher’s least significant difference (LSD) procedure. (B) Overexpression of Hu family members does not affect mRNA stability of the 6.0-kb HuR isoform. Stable HEK293 cells overexpressing the indicated constructs were treated with the transcriptional inhibitor Actinomycin D and total RNA collected from 0–8 h. HuR mRNA levels were determined via real time PCR and mean half lives were calculated via linear regression and shown ±SD from 4 independent experiments. (C) HEK293T cells were transiently transfected with equimolar amounts of firefly luciferase constructs containing the indicated portion of the HuR 3′-UTR (depicted in D) along with renilla luciferase to control for transfection efficiency. Experiments were done in the absence (Ctrl) or presence (HuR) of HuR overexpression and luciferase ratios normalized to the empty vector (pLuc) expression. Asterisks indicates P < 0.05 as determined by unpaired Student's t-test.
Figure 6. Overexpression of the neuron-specific Hu Family members (HuB, HuC and HuD) affects alternative polyadenylation of HuR mRNA. (A) Real time PCR results of transiently transfected HEK293T cells expressing the indicated FLAG tagged constructs demonstrating increased expression of alternatively polyadenylated HuR 6.0-kb mRNA, while maintaining overall HuR levels (Total). GapDH mRNA level was used as a control. Values were calculated as fold change ratios using ΔΔCT values ±SD. The means and SD's are represented from three independent experiments. Asterisks indicates P < 0.05 as determined by Fisher’s least significant difference (LSD) procedure. (B) Overexpression of Hu family members does not affect mRNA stability of the 6.0-kb HuR isoform. Stable HEK293 cells overexpressing the indicated constructs were treated with the transcriptional inhibitor Actinomycin D and total RNA collected from 0–8 h. HuR mRNA levels were determined via real time PCR and mean half lives were calculated via linear regression and shown ±SD from 4 independent experiments. (C) HEK293T cells were transiently transfected with equimolar amounts of firefly luciferase constructs containing the indicated portion of the HuR 3′-UTR (depicted in D) along with renilla luciferase to control for transfection efficiency. Experiments were done in the absence (Ctrl) or presence (HuR) of HuR overexpression and luciferase ratios normalized to the empty vector (pLuc) expression. Asterisks indicates P < 0.05 as determined by unpaired Student's t-test.CC BY-NC · PubMed Central

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Metadata

Authors
Mansfield, K. D., & Keene, J. D.
Affiliations
Duke University (US)
Corresponding author
Mansfield, Kyle D.
ORCID
Mansfield, Kyle D.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2011-12-01
Volume
40
Issue
6
Pages
2734-2746
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkr1114
PMC
PMC3315332
OpenAlex
W2091095457
Cited by
112
References
66
Citations by year
2026: 5 · 2025: 4 · 2024: 6 · 2023: 8 · 2022: 8 · 2021: 8 · 2020: 6 · 2019: 8 · 2018: 4 · 2017: 10 · 2016: 7 · 2015: 5 · 2014: 13 · 2013: 15 · 2012: 5
FWCI
4.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Science Foundation (0842621)
  • National Institutes of Health (5f32ns059100-02)
  • National Cancer Institute (R01CA157268)
  • National Institute of Neurological Disorders and Stroke (5F32NS059100-02)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
Keywords
HuR, hubs, HuC, HUD, alternative polyadenylation, neuronal differentiation, mRNA stability, 3' untranslated region, post-transcriptional regulation, RNA-binding proteins, mRNA isoforms, translation suppression
MeSH terms
Cell Line, Gene Expression Regulation, Humans, Neurons, Protein Biosynthesis, 3' Untranslated Regions, RNA Stability, Polyadenylation, ELAV Proteins, Neurogenesis, RNA Isoforms

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