KIS counteracts PTBP2 and regulates alternative exon usage in neurons
- Molecular Biology Institute of Barcelona (IBMB), CSIC, Spain
- Barts Cancer Institute, Queen Mary University of London, United Kingdom
Figures
Figure 1 with 1 supplement
KIS promotes the inclusion of exons upregulated during neuronal differentiation.
(A) Immunofluorescence staining of endogenous speckle marker SRRM2 (magenta) and 4',6-diamidino-2-phenylindole (DAPI) (bue) in HEK293T cells transfected with GFP-KIS (green). Scale bar, 2 μm. (B) …
Figure 1—figure supplement 1
KIS promotes exon inclusion in genes encoding proteins related to synaptogenesis and neurogenesis.
(A) Gene ontology (GO) term enrichment analysis of genes with more than one exon upregulated by KIS. False-discovery rate (FDR) value is shown. (B) Representative read profiles showing KIS-dependent …
Figure 2 with 1 supplement
The phosphoproteome of KIS identifies splicing regulators.
(A) MA-plot of phospho-sites found in the proteome of HEK293T cells transfected with KIS and KISK54A mutant (see also Supplementary file 3 for details). (B) STRING interaction map of top …
Figure 2—figure supplement 1
In vitro phosphorylation of PTBP2 by KIS.
(A) mRNA levels of splicing regulators from gene ontology (GO) biological process in cortical neurons at 18 DIV relative to 7 DIV (blue) or expressing shKIS relative to shCtrl (red). Mean ± standard …
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Figure 2—figure supplement 1—source data 1
KIS in vitro kinase assays.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
KIS kinase activity inhibits Ptbp2-exon-dependent exclusion.
(A) Gene enrichment analysis of PTBP2-regulated exons. Barcode plots show the position of exons excluded (red) or included (gray) by PTBP2 within the transcriptomic dataset of exons upregulated by …
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Figure 3—source data 1
CAMK2B exon exclusion analysis.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig3-data1-v2.zip
Figure 3—figure supplement 1
PTBP2 RNA-seq analysis.
(A) Gene enrichment analysis of PTBP2-regulated exons. Barcode plots show the position of exons excluded by PTBP2 within a KIS randomized exon usage dataset. Normalized enrichment scores (NES) and …
Figure 4 with 1 supplement
The functional interaction between KIS and PTBP2 has an essential role in regulating dendritic spine formation.
(A) Hippocampal neurons were co-transfected at 5 DIV with a GFP expression plasmid and either a control or PTBP2 expression plasmid. Spine morphology of transfected neurons at 14 DIV was visualized …
Figure 4—figure supplement 1
The interplay between KIS and PTBP2 has an important role in neuronal development.
(A, B) Representative confocal images of neurons from the experiment shown in Figure 4. Hippocampal neurons were transfected at 5 DIV and analyzed at 14 DIV (scale bar, 20 µm). (C) PTBP2 mRNA levels …
Figure 5 with 1 supplement
Phosphorylation by KIS disrupts PTBP2 protein complexes.
(A) Constructs expressing FLAG-PTBP2 and KIS, KISK54A were transiently transfected into HEK293T cells, and after 24 hr, cell lysates were subject to FLAG immunoprecipitation. Immunoprecipitates were …
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Figure 5—source data 1
hnRNPM and Matrin3 in FLAG-PTBP2 immunoprecipitates in vivo.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig5-data1-v2.zip
Figure 5—figure supplement 1
In vitro and in vivo experiments showing dissociation of splicing complexes by KIS kinase activity.
(A) Cell lysates from HEK293T cells expressing FLAG-PTBP2 protein were incubated with KIS recombinant proteins purified from bacteria as His fusions. FLAG immunoprecipitates were analyzed by …
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Figure 5—figure supplement 1—source data 1
hnRNPM and Matrin3 in FLAG-PTBP2 immunoprecipitates in vitro.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Representative blot of input samples from Figure 5A.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig5-figsupp1-data2-v2.zip
Figure 6 with 1 supplement
Phosphorylation by KIS compromises PTBP2 RNA-binding ability.
(A) PTBP1 and PTBP2 CLIPs were performed in HEK293T cells expressing FLAG-PTBP1 and FLAG-PTBPs proteins. UV RNA crosslinked and co-precipitated with FLAG-PTBP1 and FLAG-PTBP2s was labeled with …
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Figure 6—source data 1
PTBP2 RNA binding by CLIP.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig6-data1-v2.zip
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Figure 6—source data 2
FLAG-PTBP2 immunoprecipitated levels for CLIP analysis in Figure 6B.
- https://cdn.elifesciences.org/articles/96048/elife-96048-fig6-data2-v2.zip
Figure 6—figure supplement 1
AlphaFold2 predicts structural alterations in the triple 3SD mutants mainly affecting RRM2.
(A) AlphaFold2 prediction of the four RRM domains from wild-type (green) and 3SD (orange) PTBP2. (B) Expression variability of KIS and PTBP2 in single-cell transcriptomic data from hippocampal …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | RjOrl:SWISS | Janvier Labs | Pregnant female | |
| Cell line (H. sapiens) | HEK 293T | ATCC | CRL-11268 | From human embryo kidney tissue |
| Cell line (M. musculus) | Neuro-2a | ATCC | CCL-131 | Mouse neuroblasts isolated from brain tissue |
| Recombinant DNA reagent | pcDNA3-FLAG-PTBP1 | This work | FLAG tag fused to PTBP1 in pcDNA3 | |
| Recombinant DNA reagent | pcDNA3-FLAG-PTBP2 | This work | FLAG tag fused to PTBP2 in pcDNA3 | |
| Recombinant DNA reagent | pEGFP-C3 | Addgene | 6082-1 | N-terminal tagging vector with GFP |
| Recombinant DNA reagent | pEGFP-PTBP2 | This work | PTBP2 fused to EGFP | |
| Recombinant DNA reagent | mScarlet-C1 | Addgene | 85044 | N-terminal tagging vector with Scarlet |
| Recombinant DNA reagent | mScarlet-Matr3 | This work | Matr3 fused to Scarlet | |
| Recombinant DNA reagent | mScarlet-hnRNPM | This work | hnRNPM fused to Scarlet | |
| Recombinant DNA reagent | pGEX-KG | ATCC | 77103 | Bacterial expression vector expressing GST tag |
| Recombinant DNA reagent | pET28a | Novagen | 69864-3 | Bacterial expression vector expressing His tag |
| Recombinant DNA reagent | pcDNA3-FLAG-PTBP1 | This work | PTBP1 fused to FLAG tag | |
| Recombinant DNA reagent | pcDNA3-FLAG-PTBP2 | This work | PTBP2 fused to FLAG tag | |
| Recombinant DNA reagent | pFlareG-PSD95 exon 18 | Addgene | 90266 | Minigene reporter for alternative splicing analysis |
| Recombinant DNA reagent | pCMV-dR8.2 dvpr | Addgene | 8455 | 2nd generation lentiviral packaging plasmid |
| Recombinant DNA reagent | pCMV-VSV-G | Addgene | 8454 | Lentiviral packaging plasmid expressing envelope protein |
| Recombinant DNA reagent | pLKO.3G | Addgene | 14748 | Lentiviral plasmid expressing GFP |
| Recombinant DNA reagent | pLKO.3-DsRed shPTBP2 | This work | Lentiviral vector expressing DsRed and shRNA sequence against PTBP2 | |
| Chemical compound, drug | α-FLAG M2 (monoclonal mouse) affinity gel beads | Merck-Sigma | A2220 | |
| Antibody | α-FLAG M2 (monoclonal mouse) | Merck-Sigma | F3165 | 1:500 (WB) |
| Antibody | α-Matr3 (rabbit polyclonal) | Bionova | A300-591A-T | 1:500 (WB) |
| Antibody | α-hnRNP M3/4 (rabbit polyclonal) | Bionova | A303-910A-T | 1:500 (WB) |
| Antibody | α-mouse IRDye 680RD (goat polyclonal) | LI-COR Biosciences | 926-68070 | 1:1000 (WB) |
| Antibody | α-rabbit HRP (goat polyclonal) | Thermo Fisher | 31460 | 1:1000 (WB) |
| Antibody | α-PSD95 (mouse monoclonal) | Millipore | MABN68 | 1:400 (IF) |
| Antibody | α-SRRM2 (rabbit polyclonal) | Merck-Sigma | HPA066181 | 1:250 (IF) |
| Antibody | α-GFP Alexa 488 (rabbit polyclonal) | Thermo Fisher | A21311 | 1:1000 (IF) |
| Antibody | α-mouse Alexa 568 (donkey polyclonal) | Life Technologies | A10037 | 1:1000 (IF) |
| Antibody | α-rabbit Alexa 568 (rabbit polyclonal) | Life Technologies | A10042 | 1:1000 (IF) |
| Antibody | α-rat Alexa 568 (goat polyclonal) | Invitrogen | A11077 | 1:1000 (IF) |
| Commercial assay or kit | CalPhos mammalian transfection kit | Clontech | 631312 | |
| Commercial assay or kit | Lipofectamine 2000 | Invitrogen | 11668030 | |
| Commercial assay or kit | FuGene HD | Promega | E2311 | |
| Commercial assay or kit | NucleoSpin Plasmid Kit | Macherey-Nagel | 740588 | |
| Commercial assay or kit | NucleoBond Xtra Midi Plus EF Kit | Macherey-Nagel | 740422 | |
| Commercial assay or kit | EZNA total RNA purification Kit | Omega | R6834-01 | |
| Software, algorithm | FRETmapJ | This work | https://www.ibmb.csic.es/en/department-of-cells-and-tissues/control-of-local-mrna-expression/ | |
| Software, algorithm | SpineJ | Pedraza et al., 2014 | https://www.ibmb.csic.es/en/department-of-cells-and-tissues/control-of-local-mrna-expression/ | |
| Software, algorithm | Prism (version 9.5.1) | GraphPad | RRID: SCR_002798 | https://graphpad.com/features |
Additional files
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Supplementary file 1
RNA-seq datasets from cortical neurons.
- https://cdn.elifesciences.org/articles/96048/elife-96048-supp1-v2.xlsx
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Supplementary file 2
Phosphoproteomic data from HEK293T expressing KIS and KISK54A.
- https://cdn.elifesciences.org/articles/96048/elife-96048-supp2-v2.xlsx
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Supplementary file 3
Primers and vectors used in this study.
- https://cdn.elifesciences.org/articles/96048/elife-96048-supp3-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/96048/elife-96048-mdarchecklist1-v2.pdf
