MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA–mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells. By combining multiple complementary approaches, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation with an antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and computational analyses using bipartite and coexpression networks, we show that miRNA-mRNA interactions operate as functional modules that often correspond to cell-type identities and undergo dynamic transitions during brain development. These networks are highly dynamic during development and over the course of evolution. One such interaction is between radial-glia-enriched ORC4 and miR-2115, a great-ape-specific miRNA, which appears to control radial glia proliferation rates during human brain development.
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The data used in this study are available as part of the publicly available Gene Expression Omnibus database under accession number GSE107468.
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The authors thank S. Wang, C. Sandoval-Espinosa, E. Guzman, A. Bhaduri and N. Li for providing research resources, technical help and helpful comments during manuscript preparation. N.R. acknowledges support (Ramanujan Fellowship SB/S2/RJN-030/2017) from Science and Engineering Research Board, Department of Science & Technology, India. This research was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (K.S.K.) and National Institutes of Health (NIH) awards U54NS100717 (K.S.K.), MH105989 (A.R.K.) and R01NS075998 (A.R.K). This work was supported by a grant from the Simons Foundation (SFARI 491371; T.J.N).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Validation of the interaction between miRNA and mRNA identified by HITS-CLIP.
(a) Example of AGO2-HITS-CLIP peaks mapping to a previously validated interaction between miR-9 and the 3′ UTR of HES1 (ref. 65). (b) Distribution of HITS-CLIP reads. Several long noncoding RNAs (lncRNAs) were also identified among AGO2-bound transcripts (Supplementary Table 2), including 9 lncRNAs previously annotated as cell type specific60. (c) Luciferase reporter assay showing the down-regulation of luciferase, fused to respective genes, measured 48 h after the overexpression of respective miRNA(s) in HEK293T cells (see Methods for details). (d) Experimental design testing selected miRNA-mRNA interactions in human cells using fluorescent reporters harboring target sites identified in HITS-CLIP (same sequences as those used in c). (e) Representative images of fluorescent primary cells transfected with ORC4 MRE reporters and miRNA inhibitors. (f) Quantifications of average fluorescence change following miR inhibition relative to control (no miRNA inhibitor) for wild type reporter (wt) and a reporter lacking the MRE (mt). *- p<0.05, **- p<0.01, *** - p<0.001, **** - p<0.0001, calculated using two-tailed Student’s t-test. The number of biological replicates (individuals) is indicated above each graph. (g) We further tested the impact of nonspecific anti-miR transfections on the ORC4 WT and MT miR-2115 CDS reporter. Fluorescent reporter signal (GFP density) was normalized to loading control CAG-dsRed reporter transfection, to increase the accuracy of our analysis. The experiment was performed on three biological specimens, GW15, GW17, and GW19. We fist examined the presence of an effect using Tukey’s range test, followed by post-hoc student’s t test, P-value **- p<0.01.
(a-b) each node in mode I (target genes, a) has significantly less connections with the opposite mode than mode II (targeting miRNA, b). (c) miRNA abundance shows a long-tailed distribution. (d) Abundance of miRNA members of each module is illustrated. Modules tend to either recruit several low-abundant miRNAs or few high-abundant miRNAs.
(a-b) Both GW15-16 and GW19-20 networks illustrate a scale-free topology (Dark, median and light grey lines refer to exponential, power and truncated power law, respectively. p < 0.001). (c-d) Overlap rate matrix after hierarchical clustering for GW15-16 (c) and GW19-20 (d). For each gestational stage two replicates are produced to ensure the robustness of the community detection algorithm. (e-f) Dynamic branch cutting of panel c and d.
(a) Plot represents coefficient of variation (CV)66 for every mRNA profiled using sc-qPCR in relation to the number of HITS-CLIP reads reflecting interaction levels. Red points indicate genes not detected in HITS-CLIP. (b) Violin plots showing Pearson’s correlation coefficient between miRNA and mRNA abundance for miRNA-mRNA pairs of interactions identified by HITS-CLIP. Correlation coefficient is calculated across single cell qPCR data (Supplementary Table 7).
hybridization for miR-2115 in human cortex reveals increasing expression levels during development, reflecting sequencing results. Bottom panels, also shown in Fig. 3, show magnified view of the cortical OSVZ, scale bar is 50um.
In situ hybridization for miR-362 in human cortex reveals declining expression levels during development, reflecting sequencing results. Bottom panels, also shown in Fig. 3, show magnified view of the cortical OSVZ.
(a) Phylogeny tree of upper primates (Bonobo excluded due to poor read sequence quality in region of interest). Star represents presence of miR-2115. I) Alignment of SPINK8 intron 3 - Grey represents agreement to consensus. Black represents disagreement to consensus. Lines indicate gaps compared to consensus. II) Sequence of miR-2115 in higher primates. Highlighting indicates disagreement to consensus. miR-2115 is predicted to be present in Orangutans but is absent in Gibbons. b) Intron 3 is significantly smaller in Gibbons than higher primates (~¼ the length) and more closely matches Intron 3 of mice. There is a ~7100 nucleotide section present in Hominidae but absent in Gibbons, likely arising from an insertion occurring in Hominidae after the divergence of Gibbons. This insertion likely carried miR-2115. There is a 367 nucleotide section directly downstream of miR-2115 present in Orangutans but not Great Apes, indicating it was lost in the Great Apes common ancestor after the divergence of Orangutans. There is a 1832 nucleotide section present only in Orangutans occurring after their divergence from the Great Apes. The great mobility in this intron is likely not due to transposable elements judging from the lack of inverted repeats in the area. (c) Expression of novel miRNAs that were not annotated in latest miRBase67 across the samples. In this study, we found 36 of them expressed in prenatal brain samples. Of the expressed miRNAs, 31 of them were either human-specific or primate-specific, and 4 of them were specific expressed at GW15. Red dots indicate differentially expressed miRNAs between GW15-16 and GW19-20 by DEseq2.
In situ hybridization for miR-1286 in human prefrontal (PFC) and visual cortex (V1) reveals strong signal in the occipital cortex. Bottom panels show magnified view of the cortical OSVZ.
In situ hybridization for miR-142 in human prefrontal (PFC) and visual cortex (V1) reveals strong signal in the occipital cortex. Bottom panels show magnified view of the cortical OSVZ.
In situ hybridization for miR-548aa in human prefrontal and visual cortex reveals strong signal in the occipital cortex.
Supplementary Figure 11 Bipartite network modules are preserved between GW15-16 samples and GW19-20.
(a) Module preservation statistics shown on x-axis suggest a significant preservation of modules (modules with higher interaction levels show higher preservation as well). Modules turquoise and blue are the two least preserved modules, suggesting developmental stage-specific changes. GW15-16 module names are used to compare GW15-16 modules with their homologues in GW19-20 (see Methods for details). Module assignments are listed in Supplementary Table 6. (b) Module preservation between prenatal (rows) and adult human brain samples (columns), based on data from a HITS-CLIP analysis in the adult human cerebral cortex31,32. *-FDR<0.1, **-FDR<0.05, ***-FDR<0.01.
(a) Bipartite network module magenta is significantly enriched for genes annotated associated with ASD. (b) Expression of two miRNAs associated with ASD33 assayed using sc-qPCR expression. Notably, expression of miR-137 and miR-218 are both highly detected in neurons, with miR-218 also being expressed in progenitors. (c) In situ hybridization for miR-137 and miR-218 in primary tissue sections reveals expression of both miRNAs in the cortical plate. In particular, miR-137 appears to be expressed in maturing excitatory neurons at midgestation. ** FDR<0.05.
(a) Schematic shows mouse in vivo experimental design. Mouse cortex two days after electroporation at E13.5. White arrowhead indicates example of a dividing GFP+ radial glia cell, while yellow arrowheads indicate dividing intermediate progenitor cells. **-p<0.01, unpaired student t-test. (b) Related to Figure 4d—examines the impact of microRNA perturbation on radial glia number. We compared the proportions of SOW2 positive cells in control GFP expressing cells versus specific microRNA overexpression (miR1 or miR-2115) or specific anti-miR conditions. This analysis showed strong impact of miR-2115 on radial glia development. Interestingly, inhibition of miR-9 also lead to an increased proportion of SOX2 positive cells, consistent with previous studies68. This data was generated for three biological replicates (individuals, GW15, 17, and 19). Statistical significance was confirmed using Tukey’s test, with post hoc student t-test. P - *<0.05.
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Nowakowski, T.J., Rani, N., Golkaram, M. et al. Regulation of cell-type-specific transcriptomes by microRNA networks during human brain development. Nat Neurosci 21, 1784–1792 (2018). https://doi.org/10.1038/s41593-018-0265-3
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