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The RNA-binding protein ARPP21 controls dendritic branching by functionally opposing the miRNA it hosts

Rehfeld, Frederick, Maticzka, Daniel, Grosser, Sabine, Knauff, Pina, Eravci, Murat, Vida, Imre, Backofen, Rolf, Wulczyn, F. Gregory·2018·Nature Communications
DOI 10.1038/s41467-018-03681-3PMC PMC5964322
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Abstract

About half of mammalian miRNA genes lie within introns of protein-coding genes, yet little is known about functional interactions between miRNAs and their host genes. The intronic miRNA miR-128 regulates neuronal excitability and dendritic morphology of principal neurons during mouse cerebral cortex development. Its conserved host genes, R3hdm1 and Arpp21, are predicted RNA-binding proteins. Here we use iCLIP to characterize ARPP21 recognition of uridine-rich sequences with high specificity for 3′UTRs. ARPP21 antagonizes miR-128 activity by co-regulating a subset of miR-128 target mRNAs enriched for neurodevelopmental functions. Protein–protein interaction data and functional assays suggest that ARPP21 acts as a positive post-transcriptional regulator by interacting with the translation initiation complex eIF4F. This molecular antagonism is reflected in inverse activities during dendritogenesis: miR-128 overexpression or knockdown of ARPP21 reduces dendritic complexity; ectopic ARPP21 leads to an increase. Thus, we describe a unique example of convergent function by two products of a single gene.

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Fig. 1 miR-128 host gene conservation and ARPP21 expression during brain development. a Genomic localization of murine miR-128 within the host genes Arpp21 and R3hdm1. Two major splice isoforms of Arpp21 are depicted, as referred to in the text (Arpp21 short and long). b Domain topology of ARPP21 and R3HDM1 proteins, including major ARPP21 isoforms (Arpp21 short = ARPP21-88aa and Arpp21 long = ARPP21-807aa). c Protein sequence alignment of the core R3H motif of ARPP21 from different vertebrate species, murine R3HDM1, murine R3HDM2, and fly ENCORE using the COBALT alignment tool (NCBI). The arginine and histidine residues of the R3H motif are marked by arrows. d In situ hybridization of E15 mouse brains with probes specific for Arpp21-long and -short transcript isoforms. Expression of both isoforms is enriched in post-mitotic neurons of the cortex. The dotted line marks the lower boundary of the cortical plate. Scale bar: 100 µm. e, f Detection of ARPP21-807aa and ARPP21-88aa during brain development by western blot. e ARPP21-807aa shows a gradual increase in expression. f Expression of ARPP21-88aa is strongly increased during postnatal stages. g Immunoblot analysis of both ARPP21 protein variants in samples from different brain regions and the lymphatic system of P21 mice. ARPP21-807aa is detected in several brain regions and the thymus. ARPP21-88aa is only detected in the whole brain, cortical, striatal, and hippocampal samples. BR whole brain, CX cortex, HS hippocampus and striatum, HB hindbrain, BS brainstem, LY lymph nodes, SP spleen, TH thymus. Full western blot images are presented in Supplementary Fig. 21
Fig. 1 miR-128 host gene conservation and ARPP21 expression during brain development. a Genomic localization of murine miR-128 within the host genes Arpp21 and R3hdm1. Two major splice isoforms of Arpp21 are depicted, as referred to in the text (Arpp21 short and long). b Domain topology of ARPP21 and R3HDM1 proteins, including major ARPP21 isoforms (Arpp21 short = ARPP21-88aa and Arpp21 long = ARPP21-807aa). c Protein sequence alignment of the core R3H motif of ARPP21 from different vertebrate species, murine R3HDM1, murine R3HDM2, and fly ENCORE using the COBALT alignment tool (NCBI). The arginine and histidine residues of the R3H motif are marked by arrows. d In situ hybridization of E15 mouse brains with probes specific for Arpp21-long and -short transcript isoforms. Expression of both isoforms is enriched in post-mitotic neurons of the cortex. The dotted line marks the lower boundary of the cortical plate. Scale bar: 100 µm. e, f Detection of ARPP21-807aa and ARPP21-88aa during brain development by western blot. e ARPP21-807aa shows a gradual increase in expression. f Expression of ARPP21-88aa is strongly increased during postnatal stages. g Immunoblot analysis of both ARPP21 protein variants in samples from different brain regions and the lymphatic system of P21 mice. ARPP21-807aa is detected in several brain regions and the thymus. ARPP21-88aa is only detected in the whole brain, cortical, striatal, and hippocampal samples. BR whole brain, CX cortex, HS hippocampus and striatum, HB hindbrain, BS brainstem, LY lymph nodes, SP spleen, TH thymus. Full western blot images are presented in Supplementary Fig. 21CC BY · PubMed Central
Fig. 2 R3HDM1 and ARPP21 localize to the cytosol and show overlapping staining with the stress granule marker eIF3η in response to stress. a, b FLAG immunostainings of HeLa cells expressing N-terminally FLAG-tagged R3HDM1 or ARPP21 reveal cytosolic localization in small, perinuclear-enriched granules. The right panel in b shows a higher magnification of the dotted box in the left panel. c Diffuse, cytosolic staining of the stress granule marker eIF3η is visible under basal conditions. d Treatment of HeLa cells with arsenite induces stress granules positive for eIF3η. e, f ARPP21 and R3HDM1 colocalize with eIF3η upon arsenite stimulation. g Quantification of eIF3η colocalization with R3HDM1 and the two ARPP21 protein isoforms. Data expressed as mean ± s.d. R3HDM1: n = 94 cells. ARPP21-807aa. n = 89 cells. ARPP21-88aa: n = 141 cells. ***p < 0.001, Student’s t-test. h Schematic depiction of ARPP21 truncation mutants used in i and j. i ARPP21 lacking its C-terminal region (green) shows no colocalization with eIF3η (red) upon cellular stress. j Upon cellular stress localization of the C-terminal ARPP21 construct lacking N-terminal domains (green) overlaps with eIF3η (red). k, l Staining of endogenous ARPP21 (green), eIF3η (magenta), and phalloidin (red) in cultured primary cortical neurons after 4 days in vitro (DIV4). k Under basal conditions ARPP21 localizes to neuronal cell bodies and proximal dendrites. l Upon arsenite treatment ARPP21 colocalizes with the SG marker eIF3η. Scale bar in all images: 10 µm
Fig. 2 R3HDM1 and ARPP21 localize to the cytosol and show overlapping staining with the stress granule marker eIF3η in response to stress. a, b FLAG immunostainings of HeLa cells expressing N-terminally FLAG-tagged R3HDM1 or ARPP21 reveal cytosolic localization in small, perinuclear-enriched granules. The right panel in b shows a higher magnification of the dotted box in the left panel. c Diffuse, cytosolic staining of the stress granule marker eIF3η is visible under basal conditions. d Treatment of HeLa cells with arsenite induces stress granules positive for eIF3η. e, f ARPP21 and R3HDM1 colocalize with eIF3η upon arsenite stimulation. g Quantification of eIF3η colocalization with R3HDM1 and the two ARPP21 protein isoforms. Data expressed as mean ± s.d. R3HDM1: n = 94 cells. ARPP21-807aa. n = 89 cells. ARPP21-88aa: n = 141 cells. ***p < 0.001, Student’s t-test. h Schematic depiction of ARPP21 truncation mutants used in i and j. i ARPP21 lacking its C-terminal region (green) shows no colocalization with eIF3η (red) upon cellular stress. j Upon cellular stress localization of the C-terminal ARPP21 construct lacking N-terminal domains (green) overlaps with eIF3η (red). k, l Staining of endogenous ARPP21 (green), eIF3η (magenta), and phalloidin (red) in cultured primary cortical neurons after 4 days in vitro (DIV4). k Under basal conditions ARPP21 localizes to neuronal cell bodies and proximal dendrites. l Upon arsenite treatment ARPP21 colocalizes with the SG marker eIF3η. Scale bar in all images: 10 µmCC BY · PubMed Central
Fig. 3 ARPP21 acts as a positive regulator of post-transcriptional gene expression in a tethering assay and functionally interacts with parts of the eIF4F complex. a The MS2 coat protein was used to tether ARPP21 to the 3′ UTR of a GFP reporter transcript in 293T cells. b ARPP21 tethering results in increased GFP fluorescence of the MS2-binding site reporter (4X-MS2-BS-GFP) compared to tethering of the MS2 protein alone. Tethering of the translational inhibitor AGO2 decreases basal GFP fluorescence. Scale bar: 50 µm. c Flow cytometry analysis of tethering assays, expressed as mean fluorescent intensity (MFI) of transfected cells. AGO2-MS2 decreases, and ARPP21-MS2 and R3HDM1-MS2 increase the expression of the 4X-MS2-BS-GFP reporter, relative to Flag-MS2 alone. The C-terminal part of ARPP21 is necessary and sufficient for the positive effect on reporter expression. ***p < 0.001, Student’s t-test, n = 3, error bars represent ± s.d. d Untethered ARPP21 and R3HDM1 have no significant effect on GFP expression from the 4X-MS2-BS-GFP reporter relative to MS2 control. Statistical analysis as in c, n = 3. e FLAG-tagged ARPP21 or R3HDM1 were immunoprecipitated from 293T lysates after RNase digestion and protein interactors were identified by LC-MS/MS. f Co-immunoprecipitation (co-IP) using FLAG- or GFP-tagged ARPP21 constructs verify the ability of ARPP21 to self-interact by reciprocal co-IP. RNAse treatment strongly reduces co-IP of the RNA-binding protein IMP1 but not homomeric ARPP21 interactions. Loss of GAPDH protein after IP is shown as control. g FLAG-ARPP21 coimmunoprecipitates endogenous eIF4A and eIF4G from N2A cells. Antibodies used for detection of proteins in input and eluate are shown on the left. RNase pretreatment of lysates had no effect on co-IP of eIF4A or eIF4G. h, i ARPP21 and R3HDM1 coimmunoprecipitate eIF4A1 more efficiently than eIF4A2. HA-tagged ARPP21 or R3HDM1 was co-transfected with FLAG-tagged eIF4A1 (h) or eIF4A2 (i); results after immunoprecipitation with anti-HA antibody are shown. j Validation of endogenous eIF4G1 knockdown in 293T cells used in tethering assay. k ARPP21 activity in the tethering assay is reduced after eIF4G knockdown. ***p < 0.001, two-way ANOVA with Bonferroni post-test, n = 4, error bars represent s.d. Full western blot images are presented in Supplementary Fig. 21
Fig. 3 ARPP21 acts as a positive regulator of post-transcriptional gene expression in a tethering assay and functionally interacts with parts of the eIF4F complex. a The MS2 coat protein was used to tether ARPP21 to the 3′ UTR of a GFP reporter transcript in 293T cells. b ARPP21 tethering results in increased GFP fluorescence of the MS2-binding site reporter (4X-MS2-BS-GFP) compared to tethering of the MS2 protein alone. Tethering of the translational inhibitor AGO2 decreases basal GFP fluorescence. Scale bar: 50 µm. c Flow cytometry analysis of tethering assays, expressed as mean fluorescent intensity (MFI) of transfected cells. AGO2-MS2 decreases, and ARPP21-MS2 and R3HDM1-MS2 increase the expression of the 4X-MS2-BS-GFP reporter, relative to Flag-MS2 alone. The C-terminal part of ARPP21 is necessary and sufficient for the positive effect on reporter expression. ***p < 0.001, Student’s t-test, n = 3, error bars represent ± s.d. d Untethered ARPP21 and R3HDM1 have no significant effect on GFP expression from the 4X-MS2-BS-GFP reporter relative to MS2 control. Statistical analysis as in c, n = 3. e FLAG-tagged ARPP21 or R3HDM1 were immunoprecipitated from 293T lysates after RNase digestion and protein interactors were identified by LC-MS/MS. f Co-immunoprecipitation (co-IP) using FLAG- or GFP-tagged ARPP21 constructs verify the ability of ARPP21 to self-interact by reciprocal co-IP. RNAse treatment strongly reduces co-IP of the RNA-binding protein IMP1 but not homomeric ARPP21 interactions. Loss of GAPDH protein after IP is shown as control. g FLAG-ARPP21 coimmunoprecipitates endogenous eIF4A and eIF4G from N2A cells. Antibodies used for detection of proteins in input and eluate are shown on the left. RNase pretreatment of lysates had no effect on co-IP of eIF4A or eIF4G. h, i ARPP21 and R3HDM1 coimmunoprecipitate eIF4A1 more efficiently than eIF4A2. HA-tagged ARPP21 or R3HDM1 was co-transfected with FLAG-tagged eIF4A1 (h) or eIF4A2 (i); results after immunoprecipitation with anti-HA antibody are shown. j Validation of endogenous eIF4G1 knockdown in 293T cells used in tethering assay. k ARPP21 activity in the tethering assay is reduced after eIF4G knockdown. ***p < 0.001, two-way ANOVA with Bonferroni post-test, n = 4, error bars represent s.d. Full western blot images are presented in Supplementary Fig. 21CC BY · PubMed Central
Fig. 4 iCLIP identifies the ARPP21–RNA interactome. a ARPP21 deletion constructs used in UV-crosslinking experiments to assess their RNA-binding potential. Construct 2 conforms to a splice variant that lacks a part of the SUZ domain. b SDS-PAGE with FLAG immunoblot confirms immunoprecipitation of all ARPP21 variants, lane designations refer to constructs depicted in a. c Autoradiogramm of crosslinked and 32P-labeled ARPP21–RNA complexes separated by NuPAGE, lane designations as in a and b. All variants harboring at least one of the predicted RNA-binding domains exhibit RNA-binding ability (*), the C-terminal only Construct 6 yielded no signal. d Schematic overview of the iCLIP procedure performed with iARPP21 cells. Unique barcodes (blue) introduced during the reverse transcription allow for multiplexing iCLIP samples. Due to the circularization step, truncated cDNAs are captured and the position of protein–RNA crosslinking (red) can be accurately assessed. Green: deep-sequencing adapters. e Representative ARPP21 iCLIP coverage at the Phf6 locus shows specificity for the 3′UTR. The negative control iCLIP from cells not expressing ARPP21 shows negligible background. Numerical scale on left refers to unique crosslinking events (CEs). f Genome-wide characterization of unique CEs for ARPP21 by RNA type. g 3′UTRs of ARPP21 targets are longer compared to non-ARPP21 target transcripts. Median 3′UTR length of ARPP21 target mRNA: 1.99 kb. Median 3′UTR length of ARPP21-unbound mRNA: 0.72 kb. h Nucleotide representation flanking the crosslink site as determined by GraphProt identifies a uridine-rich motif. i ARPP21 iCLIP signals are enriched at uridine-rich sequences. A representative peak from the Phf6 3′UTR is shown; the numerical scale at left refers to unique CEs. j Secondary structure analysis of the ARPP21 interaction motif by GraphProt. The center of the motif containing the crosslinked nucleotide is largely devoid of predicted stems. E external loop, H hairpin, I internal loop, M multi loop, S stem
Fig. 4 iCLIP identifies the ARPP21–RNA interactome. a ARPP21 deletion constructs used in UV-crosslinking experiments to assess their RNA-binding potential. Construct 2 conforms to a splice variant that lacks a part of the SUZ domain. b SDS-PAGE with FLAG immunoblot confirms immunoprecipitation of all ARPP21 variants, lane designations refer to constructs depicted in a. c Autoradiogramm of crosslinked and 32P-labeled ARPP21–RNA complexes separated by NuPAGE, lane designations as in a and b. All variants harboring at least one of the predicted RNA-binding domains exhibit RNA-binding ability (*), the C-terminal only Construct 6 yielded no signal. d Schematic overview of the iCLIP procedure performed with iARPP21 cells. Unique barcodes (blue) introduced during the reverse transcription allow for multiplexing iCLIP samples. Due to the circularization step, truncated cDNAs are captured and the position of protein–RNA crosslinking (red) can be accurately assessed. Green: deep-sequencing adapters. e Representative ARPP21 iCLIP coverage at the Phf6 locus shows specificity for the 3′UTR. The negative control iCLIP from cells not expressing ARPP21 shows negligible background. Numerical scale on left refers to unique crosslinking events (CEs). f Genome-wide characterization of unique CEs for ARPP21 by RNA type. g 3′UTRs of ARPP21 targets are longer compared to non-ARPP21 target transcripts. Median 3′UTR length of ARPP21 target mRNA: 1.99 kb. Median 3′UTR length of ARPP21-unbound mRNA: 0.72 kb. h Nucleotide representation flanking the crosslink site as determined by GraphProt identifies a uridine-rich motif. i ARPP21 iCLIP signals are enriched at uridine-rich sequences. A representative peak from the Phf6 3′UTR is shown; the numerical scale at left refers to unique CEs. j Secondary structure analysis of the ARPP21 interaction motif by GraphProt. The center of the motif containing the crosslinked nucleotide is largely devoid of predicted stems. E external loop, H hairpin, I internal loop, M multi loop, S stemCC BY · PubMed Central
Fig. 5 ARPP21 and miR-128 bind an overlapping set of target mRNAs with related functions. a Venn diagram of conserved TargetScan 7.1 miR-128 target mRNAs, and ARPP21 iCLIP substrate mRNAs. b 7mer-A1 seed match occurrence per 3′UTR of ARPP21 targets for several conserved miRNAs. c Number of occurrences of miR-128 seed matches and poly-uridine (poly-U) 7mers per 3′UTR among ARPP21-bound and ARPP21-unbound transcripts. Both motifs are significantly enriched in 3′UTRs of ARPP21 target mRNAs. Welch two-sample t-test, ***p < 2.2 × 10−16. d miR-128 and poly-U 7mer occurrence normalized per kilobase of 3′UTR comparing ARPP21 target and non-target transcripts. ***p < 2.2 × 10−16
Fig. 5 ARPP21 and miR-128 bind an overlapping set of target mRNAs with related functions. a Venn diagram of conserved TargetScan 7.1 miR-128 target mRNAs, and ARPP21 iCLIP substrate mRNAs. b 7mer-A1 seed match occurrence per 3′UTR of ARPP21 targets for several conserved miRNAs. c Number of occurrences of miR-128 seed matches and poly-uridine (poly-U) 7mers per 3′UTR among ARPP21-bound and ARPP21-unbound transcripts. Both motifs are significantly enriched in 3′UTRs of ARPP21 target mRNAs. Welch two-sample t-test, ***p < 2.2 × 10−16. d miR-128 and poly-U 7mer occurrence normalized per kilobase of 3′UTR comparing ARPP21 target and non-target transcripts. ***p < 2.2 × 10−16CC BY · PubMed Central
Fig. 6 ARPP21 and miR-128 have antagonistic functions on an overlapping set of target mRNAs. a, b Conserved TargetScan predictions for miR-128-binding sites (orange bars) and ARPP21 iCLIP CL signals (turquoise) at the 3′UTRs of the miR-128 target mRNAs Phf6 and Msk1. c Schematic of 3′UTR GFP-reporter assay to assess direct effects of miR-128 and ARPP21 on individual transcripts. d, e Phf6 and Msk1 3′UTR reporter fluorescence upon miR-128 and/or ARPP21 expression normalized to control transfection. miR-128 represses and ARPP21 increases GFP reporter expression; co-transfection leads to intermediate expression. Data expressed as mean ± s.d., ***p < 0.001, Student’s t-test, n = 3. f Representative immunoblot analysis of endogenous PHF6 and MSK1 protein levels in 293T cells. ARPP21 transfection leads to increased protein expression of MSK1 and PHF6. miR-128 mimic transfection results in reduced expression of MSK1 and PHF6 protein. Co-transfection leads to intermediate levels. g, h Quantification of PHF6 and MSK1 protein expression from five independent experiments as in f. *p < 0.05, ***p < 0.001, one-sample t-test against 100%, data represents mean ± s.d. i miR-128 overexpression by lentivirus in primary cortical neurons at DIV7 reduces MSK1 and PHF6 protein expression. The arrowhead marks the specific PHF6 band. j Quantification of miR-128 overexpression effect on MSK1 and PHF6 protein levels. One-sample t-test against 100%, n = 4 biological replicates, *p < 0.05; **p < 0.01. Data expressed as mean ± s.d. Protein levels of ARPP21, MSK1, and PHF6 upon ARPP21 overexpression (k) and knockdown (l) in primary cortical neurons at DIV7. Quantification of protein expression upon overexpression (m) or knockdown (n) of ARPP21. One-sample t-test against 100%, n = 6 biological replicates, *p < 0.05. Data expressed as mean ± s.d. Full western blot images are presented in Supplementary Fig. 21
Fig. 6 ARPP21 and miR-128 have antagonistic functions on an overlapping set of target mRNAs. a, b Conserved TargetScan predictions for miR-128-binding sites (orange bars) and ARPP21 iCLIP CL signals (turquoise) at the 3′UTRs of the miR-128 target mRNAs Phf6 and Msk1. c Schematic of 3′UTR GFP-reporter assay to assess direct effects of miR-128 and ARPP21 on individual transcripts. d, e Phf6 and Msk1 3′UTR reporter fluorescence upon miR-128 and/or ARPP21 expression normalized to control transfection. miR-128 represses and ARPP21 increases GFP reporter expression; co-transfection leads to intermediate expression. Data expressed as mean ± s.d., ***p < 0.001, Student’s t-test, n = 3. f Representative immunoblot analysis of endogenous PHF6 and MSK1 protein levels in 293T cells. ARPP21 transfection leads to increased protein expression of MSK1 and PHF6. miR-128 mimic transfection results in reduced expression of MSK1 and PHF6 protein. Co-transfection leads to intermediate levels. g, h Quantification of PHF6 and MSK1 protein expression from five independent experiments as in f. *p < 0.05, ***p < 0.001, one-sample t-test against 100%, data represents mean ± s.d. i miR-128 overexpression by lentivirus in primary cortical neurons at DIV7 reduces MSK1 and PHF6 protein expression. The arrowhead marks the specific PHF6 band. j Quantification of miR-128 overexpression effect on MSK1 and PHF6 protein levels. One-sample t-test against 100%, n = 4 biological replicates, *p < 0.05; **p < 0.01. Data expressed as mean ± s.d. Protein levels of ARPP21, MSK1, and PHF6 upon ARPP21 overexpression (k) and knockdown (l) in primary cortical neurons at DIV7. Quantification of protein expression upon overexpression (m) or knockdown (n) of ARPP21. One-sample t-test against 100%, n = 6 biological replicates, *p < 0.05. Data expressed as mean ± s.d. Full western blot images are presented in Supplementary Fig. 21CC BY · PubMed Central
Fig. 7 Phenotype of ARPP21 loss- and gain-of-function during cortical neuron differentiation in vivo. a IUE at E15.5 with Arpp21 shRNA or overexpression constructs followed by morphological analysis at P14. b qRT-PCR of endogenous Arpp21 mRNA levels in N2A cells confirms efficacy of Arpp21 shRNA knockdown using Oaz1 mRNA as standard. ***p < 0.001, Student’s t-test, n = 3. Data represent mean ± s.d. c Representative reconstructions of a neuron expressing a non-targeting control shRNA (shNeg) and an ARPP21 knockdown (shArpp21) neuron. Scale bar: 50 µm. d Sholl analysis of control (shNeg; black) and ARPP21 knockdown (shArpp21; turquoise)-expressing neurons shows significantly decreased dendritic complexity upon ARPP21 knockdown. e Reconstructions of representative neurons expressing either GFP or ARPP21 under control of the NeuroD1 (ND1) promoter (plasmids ND1::IRES-GFP or ND1::ARPP21-IRES-GFP, respectively). f Sholl analysis reveals a significantly increased dendritic complexity of ARPP21-overexpressing (ND1::Arpp21; turquoise) neurons compared to control neurons (ND1::GFP; black). *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni post-test. shNeg: n = 14 cells. shArpp21: n = 11 cells. ND1-GFP: n = 20 cells. ND1-Arpp21: n = 14 cells. Data expressed as mean ± s.e.m. Scale bar: 50 µm
Fig. 7 Phenotype of ARPP21 loss- and gain-of-function during cortical neuron differentiation in vivo. a IUE at E15.5 with Arpp21 shRNA or overexpression constructs followed by morphological analysis at P14. b qRT-PCR of endogenous Arpp21 mRNA levels in N2A cells confirms efficacy of Arpp21 shRNA knockdown using Oaz1 mRNA as standard. ***p < 0.001, Student’s t-test, n = 3. Data represent mean ± s.d. c Representative reconstructions of a neuron expressing a non-targeting control shRNA (shNeg) and an ARPP21 knockdown (shArpp21) neuron. Scale bar: 50 µm. d Sholl analysis of control (shNeg; black) and ARPP21 knockdown (shArpp21; turquoise)-expressing neurons shows significantly decreased dendritic complexity upon ARPP21 knockdown. e Reconstructions of representative neurons expressing either GFP or ARPP21 under control of the NeuroD1 (ND1) promoter (plasmids ND1::IRES-GFP or ND1::ARPP21-IRES-GFP, respectively). f Sholl analysis reveals a significantly increased dendritic complexity of ARPP21-overexpressing (ND1::Arpp21; turquoise) neurons compared to control neurons (ND1::GFP; black). *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni post-test. shNeg: n = 14 cells. shArpp21: n = 11 cells. ND1-GFP: n = 20 cells. ND1-Arpp21: n = 14 cells. Data expressed as mean ± s.e.m. Scale bar: 50 µmCC BY · PubMed Central
Fig. 8 Schematic depiction of our model for the miR-128-ARPP21 regulatory circuit and its impact on the post-transcriptional regulation of common mRNA targets. a ARPP21 and miR-128 are co-expressed from the same genetic locus. pri-miR-128-2 is generated upon Arpp21 transcription and subsequently processed by Drosha and Dicer into mature miR-128. miR-128-loaded miRISC binds and downregulates target mRNAs that possess the miR-128 seed match sequence. Arpp21 is transcribed, spliced, and translated into protein. ARPP21 binds a subset of miR-128 target mRNAs via a uridine-rich sequence motif leading to increased protein expression. Mechanisms that could potentially regulate the relative activities of ARPP21 and miR-128 are marked in red. Alternative splicing of the Arpp21 mRNA and phosphorylation of ARPP21 protein could regulate its activity. Further, miR-128 can inhibit Arpp21 expression through a conserved seed match in the Arpp21 3′UTR. On the other hand, miR-128 processing is regulated during development. b Co-regulated transcripts have a greater dynamic range of gene expression compared to transcripts under control of either miR-128 or ARPP21 acting on their own. Dendritic complexity of cortical neurons is highly sensitive to the relative levels of miR-128 and ARPP21
Fig. 8 Schematic depiction of our model for the miR-128-ARPP21 regulatory circuit and its impact on the post-transcriptional regulation of common mRNA targets. a ARPP21 and miR-128 are co-expressed from the same genetic locus. pri-miR-128-2 is generated upon Arpp21 transcription and subsequently processed by Drosha and Dicer into mature miR-128. miR-128-loaded miRISC binds and downregulates target mRNAs that possess the miR-128 seed match sequence. Arpp21 is transcribed, spliced, and translated into protein. ARPP21 binds a subset of miR-128 target mRNAs via a uridine-rich sequence motif leading to increased protein expression. Mechanisms that could potentially regulate the relative activities of ARPP21 and miR-128 are marked in red. Alternative splicing of the Arpp21 mRNA and phosphorylation of ARPP21 protein could regulate its activity. Further, miR-128 can inhibit Arpp21 expression through a conserved seed match in the Arpp21 3′UTR. On the other hand, miR-128 processing is regulated during development. b Co-regulated transcripts have a greater dynamic range of gene expression compared to transcripts under control of either miR-128 or ARPP21 acting on their own. Dendritic complexity of cortical neurons is highly sensitive to the relative levels of miR-128 and ARPP21CC BY · PubMed Central

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Metadata

Authors
Rehfeld, F., Maticzka, D., Grosser, S., Knauff, P., Eravci, M., Vida, I., Backofen, R., & Wulczyn, F. G.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-03-26
Volume
9
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-018-03681-3
PMC
PMC5964322
Cited by
74
References
8
Language
en
Licence

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