5'-UTR SNP of FGF13 causes translational defect and intellectual disability
- Institute of Neuroscience and State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
- Shanghai Brain-Intelligence Project Center, China
- Shanghai Clinical Center, Chinese Academy of Sciences/Xu-Hui Central Hospital, China
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China
- School of Life Science and Technology, Shanghai Tech University, China
- CAS-MPG Partner Institute for Computational Biology, Chinese Academy of Sciences, China
- State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China
- Shanghai Advanced Research Institute, Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
The newly identified single-nucleotide polymorphism (SNP) in FGF13 impairs the protein translation.
(A) The number of subjects involved in the present study is listed. A sum of 100 severe intellectual disability (ID) children (intelligence quotient [IQ] < 40) with detailed clinical histories were …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Manipulation of the FGF13 5′-untranslated region (5′-UTR) in induced pluripotent stem cells (iPSCs) with the CRISPR/Cas9 editing.
(A) The expression of FGF13 mRNA is shown through reanalysis of single-cell data from the human fetal prefrontal cortex (Zhong et al., 2018). The abundance of FGF13 mRNA in the neuron and neural …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Mice with the point mutation in Fgf13 5′-untranslated region (5′-UTR) reduce protein translation and exhibit impaired learning and memory.
(A) The region around the mutation site is highly conserved among species. The mutation site is highlighted with the arrow and remains constant in various species. (B) The homology point mutation in …
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Construction of mice with the point mutation in Fgf13 5′-untranslated region (5′-UTR) using CRISPR/Cas9 system.
(A) The FGF13 mRNA level was not changed in HEK293 cells expressing the mutant (Mut) 5′-UTR of Fgf13 compared with that of the wildtype (WT) one (n = 5, p=0.3489 and p=0.2096 for isoforms 2 and 4). …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
Fgf13 point mutation mice show delayed radial migration and increased axon branching.
(A, B) Representative images and quantitative analysis of the cerebral cortex at P0 in Fgf13 point mutant (Mut) mice electroporated with pcDNA3.1 vector at E14.5 showed that the radial migration of …
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Alterations of neuronal migration, axon projection, and spine density in the brain of Fgf13 5′-untranslated region (5′-UTR) mutant (Mut) mice.
(A, B) The representative images of the cerebral cortex of Fgf13 5′-UTR Mut mice at P14, electroporated with the plasmid expressing the green fluorescent protein (GFP) at E14.5, show that the radial …
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
The 5′-untranslated region (5′-UTR) point mutation impairs association of FGF13 mRNA with ribosomes.
(A) The overall mRNA levels in the brain of wildtype (WT) or mutant (Mut) mouse revealed by UV absorption profiles (254 nm). A total of 16 fractions were collected from the sucrose density …
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
The association of FGF13 mRNA with heavy polysomes is decreased in Fgf13 5′-untranslated region (5′UTR) mutant mice.
(A) Polysome profiles from brains of six wildtype (WT) and six mutant (Mut) mice (for analysis in Figure 4C, D) are shown. The distribution of FGF13 mRNA and GAPDH mRNA in different fractions was …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 1 supplement
The 5′-untranslated region (5'-UTR) point mutation impairs the binding of PTBP1/2 with FGF13 5′-UTR.
(A) Schematic graph shows the procedure of RNA pull-down assay. Biotin-labeled synthesized RNA was perfused with cell extracts or purified protein and enriched in streptavidin magnetic beads that …
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
The FGF13 5′-untranslated region (5′-UTR) single-nucleotide polymorphism (SNP) does not induce the alteration of internal ribosome entry site (IRES) entry and mRNA structure.
(A) Dual-luciferase assay was used to test the IRES activity of FGF13 5′-UTR. The known IRES sequence (EMCV) in the pRIF plasmid was used as a positive control, and the wildtype (WT) or mutant (Mut) …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Enhancement of the PTBP2 binding with mutant (Mut) Fgf13 5′-untranslated region (5′-UTR) rescues the impairment of neuronal morphogenesis.
(A) Immunoblotting showed that the PTBP1 knockdown reduced the translation of co-expressed human FGF13 isoform 2 in HEK293 cells (n = 4, p=0.0077 for FGF13, p=0.0032 for PTBP1). The mRNA level of …
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/63021/elife-63021-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
The subcellular distribution of PTBP2 in mouse cortical neurons at DIV4.
Immunoblotting with antibody against PTBP2 detected bands in both nuclear and cytoplasmic components, which are marked by Lamin-B1 and GAPDH, respectively.
Figure 7
A proposed model of the FGF13 5′-untranslated region (5′-UTR) single-nucleotide polymorphism (SNP) in attenuating protein translation and impairing brain development.
In normal situation, PTBP2 binds to the 5′-UTR of FGF13 and recruits the ribosomes to initiate the protein translation. However, the C>G mutation in the FGF13 5′-UTR of intellectual disability (ID) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) male and female | Fgf13 mutant | This paper | Home-made | Obtained by contacting the correspondence author |
| Antibody | Anti-FGF13(goat polyclonal) | Santa Cruz | Cat# sc-16811 RRID:AB_2104044 | IB (1:500) |
| Antibody | Anti-FGF13(rabbit polyclonal) | Wu et al., 2012 | Home-made by immunization with FGF13B 182–192 aa | IB (1:5000) |
| Antibody | Anti-HA(mouse monoclonal) | Sigma | Cat# H3663 RRID:AB_262051 | IB (1:2000); IP (1 μg) |
| Antibody | Anti-Flag(mouse monoclonal) | Sigma | Cat# F3165 RRID:AB_259529 | IB (1:10,000) |
| Antibody | Anti-actin(mouse monoclonal) | Chemicon | Cat# MAB1501 RRID:AB_2223041 | IB (1:400,000) |
| Antibody | Anti-MATR3(rabbit monoclonal) | Abcam | Cat# ab151739 RRID:AB_2885091 | IB (1:2000) |
| Antibody | Anti-PTBP1(mouse monoclonal) | This paper | Home-made from ATCC Cat# CRL-2501 | Source: RRID:CVCL_L596; IB (1:2000) |
| Antibody | Anti-PTBP2(mouse monoclonal) | Abcam | Cat# ab57619 RRID:AB_2284865 | IB (1:500); IP (4 μg) |
| Antibody | Anti-Lamin B1(mouse monoclonal) | Proteintech | Cat# 66095-1-Ig RRID:AB_2721256 | IB (1:5000) |
| Antibody | Anti-SOX2(rabbit polyclonal) | Stem Cell Technologies | Cat# 60055 RRID:N/A | ICC (1:100) |
| Antibody | Anti-Tuj1(mouse monoclonal) | Chemicon | Cat# CBL412 RRID:AB_11205398 | ICC (1:1000) |
| Antibody | Anti-Tuj1(chicken polyclonal) | Abcam | Cat# ab107216 RRID:AB_10899689 | ICC (1:1000) |
| Antibody | Anti-GFP(chicken polyclonal) | Abcam | Cat# ab13970 RRID:AB_300798 | ICC/IHC (1:2000) |
| Antibody | Anti-SMI-312(mouse monoclonal) | BioLegend | Cat# 837904 RRID:AB_2566782 | ICC (1:1000) |
| Antibody | Anti-Cux1(rabbit polyclonal) | Santa Cruz | Cat# sc-13024 RRID:AB_2261231 | IHC (1:1000) |
| Recombinant DNA reagent | Plasmids | This paper | Supplementary file 6 | |
| Sequence-based reagent | Oligonucleotides, primers | This paper | Supplementary file 5 | |
| IB: immunoblotting; IP: immunoprecipitation; IHC: immunohistochemistry; ICC: immunocytochemistry. | ||||
Additional files
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Supplementary file 1
List of 262 reported intellectual disability-related genes from database and literature screening.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp1-v1.docx
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Supplementary file 2
List of single-nucleotide polymorphisms and insertions and deletions of three families from exon-capture-sequencing.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp2-v1.docx
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Supplementary file 3
List of single-nucleotide polymorphisms, insertions and deletions, and copy number variations of three children from whole-genome sequencing.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp3-v1.docx
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Supplementary file 4
Clinical records of three intellectual disability individuals.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp4-v1.docx
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Supplementary file 5
Primers and oligonucleotides used for real-time PCR, Cas9 targeting, sequencing, plasmid construction, and biotin probe.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp5-v1.docx
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Supplementary file 6
Antibodies, reagents, cell lines, plasmid constructs, software, and other resources used in this study.
- https://cdn.elifesciences.org/articles/63021/elife-63021-supp6-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/63021/elife-63021-transrepform-v1.docx
