MicroRNA-934 is a novel primate-specific small non-coding RNA with neurogenic function during early development
- Laboratory of Cellular and Molecular Neurobiology-Stem Cells, Department of Neurobiology, Hellenic Pasteur Institute, Greece
- Department of Pathology, Beth Israel Deaconess Medical Center, United States
- DIANA-Lab, Hellenic Pasteur Institute, Greece
- Harvard Medical School, United States
- Broad Institute of MIT and Harvard, United States
Figures
Figure 1
Directed neural differentiation of hESC/iPSC and RNA/small RNA sequencing at distinct stages of differentiation.
(A) Schematic representation of directed neural differentiation of hESCs/iPSCs. Embryoid body (EB) formation (5–6 DIV) is followed by dual SMAD inhibition for neural induction and NPC generation …
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Figure 1—source data 1
qRT PCR data for PAX6 and DCX relative expression.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig1-data1-v2.xlsx
Figure 2
miR-934 is specifically expressed during progenitor expansion and early neuron generation.
(A) Heatmap profiling the expression of selected characteristic miRNAs across directed neural differentiation of hESCs/iPSCs to NPCs and neurons, demonstrating also the high expression of miR-934 …
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Figure 2—source data 1
qRT PCR data for hsa-miR-934 relative expression.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig2-data1-v2.xlsx
Figure 3
Perturbation of miR-934 affects the balance between Pax6+ progenitors and DCX+ neuroblasts and impacts on the morphological transition of newly born neurons.
(A–H) Representative confocal images of hESC-derived cultures at neural induction stage (DIV12) following immunolabeling for Pax6 (green) and Nestin (red) or for doublecortin (DCX, green), upon …
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Figure 3—source data 1
Data on estimation of % Relative PAX+ and DCX+ cells.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig3-data1-v2.xlsx
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Figure 3—source data 2
Data on measurements of Longest Process in TUJ1+ neurons and of % of bipolar TUJ1+ neurons.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig3-data2-v2.xlsx
Figure 4
Identification of miR-934 targets during neural induction.
(A) Confocal image showing PAX6+ neural progenitors (red) at DIV 15 transduced with miRZip lentivector (GFP fluorescence, green) for sustained inhibition of miR-934. Scale bar: 20 μm. (B) qRT-PCR …
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Figure 4—source data 1
qRT PCR data for expression of miR-934 predicted targets upon sustained miR934 inhibition.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig4-data1-v2.xlsx
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Figure 4—source data 2
Data on luciferase activity in HEK293T cells upon co-transfection with miR-934 mimics and the MRE (8-mer binding site) reporter construct.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig4-data2-v2.xlsx
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Figure 4—source data 3
Data on western blot results for FZD5 and b-catenin proteins following miR934 Sustained inhibition.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig4-data3-v2.xlsx
Figure 5
Sustained Inhibition of miR-934 induces global expression changes in genes associated with NPC proliferation and neuronal differentiation.
(A) GO enrichment analysis of differential gene expression data following sustained inhibition of miR-934. (B–E) Heatmaps presenting the log2-fold change of molecules involved in the indicated …
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Figure 5—source data 1
qRT PCR data for expression of genes affected by miR-934 inhibition.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig5-data1-v2.xlsx
Figure 6
miRNA expression profiling following sustained inhibition of miR-934.
(A) Heatmap showing the 20 miRNAs with lowest p-values at differential expression analysis following miR-934 sustained inhibition. (B) Table describing the association of differentially expressed …
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Figure 6—source data 1
qRT-PCR data for expression of miR-34c-5p following miR-934 sustained inhibition.
- https://cdn.elifesciences.org/articles/50561/elife-50561-fig6-data1-v2.xlsx
Appendix 1—figure 1
Graph incorporating miRNA expression data from uniformly-analyzed small RNA-Seq libraries of non-neural human tissue (detailed in Supplementary file 3).
Association was noted only in one case (breast).
Appendix 1—figure 2
Identification of binding sites for miR-934 on the 3'-UTR of TFCP2L1 and RAB3B.
(A) Schematic representation of TFCP2L1 3’-UTR depicting in red fonts the seed-binding sequences of the two predicted MREs for miR-934 with their respective positions. To examine mechanistically the …
Appendix 1—figure 3
Identification of an 8mer-seed sequence as binding site for miR-934 on the 3'-UTR of Fzd5.
(A) RT-qPCR analysis at different time points of neural induction, showing up-regulation of miR-934 and parallel down-regulation of the mRNA of its predicted target, Fzd5. Using the DIANA-microT-CDS …
Appendix 1—figure 4
Heatmaps showing the expression of genes displaying no significant differences following miR-934 sustained inhibition.
(A) Genes associated with intermediate progenitors, outer radial glia and cortical plate neurons. (B) Genes associated with regional identities during brain development.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | hESC line HUES6 | Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, et al. Derivation of embryonic stem-cell lines from human blastocysts. The New England journal of medicine. 2004;350 (Muñoz-Sanjuán and Brivanlou, 2002):1353–6. | RRID:CVCL_B19 | Cell line (H. sapiens) |
| Chemical compound, drug | Knockout Serum Replacement (KSR) | Life Technologies Thermo Fisher | Life Technologies, Thermo Fisher 10828–010 | |
| Chemical compound, drug | KnockOut DMEM medium | Life Technologies Thermo Fisher | Life Technologies, Thermo Fisher 10829–018 | |
| Chemical compound, drug | matrigel matrix | BD biosciences | BD 354234 | See Materials and methods |
| Chemical compound, drug | Noggin | R and D Systems | R and D 6057 NG-25 | See Materials and methods |
| Chemical compound, drug | SB431542 | Tocris Biosciences | Tocris Biosciences 1614 | See Materials and methods |
| Commercial assay or kit | RNA Clean and Concentrator kit | Zymo Research | Zymo Research R1018 | |
| Recombinant DNA reagent | pmiR-GLO reporter vector | Promega | Promega E1330 | |
| Recombinant DNA reagent | miRZip lentivector-based anti-microRNA system | Systems Biosciences | https://systembio.com/products/mirna-and-lncrna-research-tools/mirzip-knockdown-vectors/mirzip-anti-mirna-constructs/ | |
| Chemical compound, drug | Lipofectamine 2000 | Life Technologies/Thermo | Life Technologies11668–019 | |
| Sequenced-based reagent | miR-934 mimics | Exiqon/Qiagen | http://www.exiqon.com/ls/Pages/ProductDetails.aspx?ProductId=k1WVIuhVVQH848C13pCVyN5WGs82ItaE%2flSNAGCdK1%2bEWkr83WchdvRAbgzMNpIk3WDIx9O1eBJZUcYGe81LzgTaw2jJcGjXgRYNy58buck%3d&CategoryType=2yRAQGj8eprRK3%2bx8lE0zg%3d%3d | |
| Sequenced-based reagent | miR-934 inhibitors | Dharmacon | https://horizondiscovery.com/products/gene-modulation/knockdown-reagents/mirna/PIFs/miRIDIAN-microRNA-Hairpin-Inhibitor?nodeid=mirnamature-mimat0004977 | |
| Antibody | mouse monoclonal anti-PAX6 | Developmental Studies Hybridoma Bank | RRID:AB_528427 | Dilution (1:50) See Materials and methods |
| Antibody | goat polyclonal anti-doublecortin (DCX) | Santa Cruz | Sc-8067 | Dilution (1:100) See Materials and methods |
| Antibody | rabbit polyclonal anti-Frizzled 5 | Abcam | Ab75234 | Dilution (1:1000) See Materials and methods |
| Antibody | rabbit monoclonal anti-active β-catenin | Cell signaling | Cell signaling, 19807 | Dilution (1:1000) See Materials and methods |
| Software, algorithm | ImageJ | ImageJ (http://imagej.nih.gov/ij/) | RRID:SCR_003070 | |
| Sequenced-based reagent | RT-qPCR primers | This paper | See Supplementary file 7 |
Additional files
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Supplementary file 1
Supplemental table 1 showing the 144 miRNAs across all differentiation stages demonstrating tissue specificity index tau >0.7.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp1-v2.docx
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Supplementary file 2
Supplemental table 2 presenting The 10 most highly expressed miRNAs in NPCs demonstrating tissue specificity index tau >0.7.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp2-v2.docx
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Supplementary file 3
Supplemental table 3 showing All Blastn results with evalue <1 for a query of the mature hsa-miR-934 sequence against all mature miRNAs in miRBase v22.1.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp3-v2.docx
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Supplementary file 4
Supplemental table 4 presenting IDs and description of the small RNA-Seq human libraries of human tissue and cell data included in the analysis for miR934 expression.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp4-v2.docx
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Supplementary file 5
Supplemental table 5 showing all the predicted mRNA targets for miRNA-934.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp5-v2.docx
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Supplementary file 6
Supplemental table 6 presenting the 3’ UTR binding sites for miR934 on its four identified targets (adapted from Diana microT-CDS).
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp6-v2.docx
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Supplementary file 7
Supplemental table 7 listing the sequences of primers and oligos used in this study.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp7-v2.docx
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Supplementary file 8
Supplemental table 8 showing information on progenitor-specific and SP-enriched genes affected by sustained inhibition of miR-934.
- https://cdn.elifesciences.org/articles/50561/elife-50561-supp8-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50561/elife-50561-transrepform-v2.pdf
