Article Figures & Data
Figures
- Figure 1. Pre-miRNAs expressed in the CNS structures as revealed by RNA-Seq.
(A) Pie chart of miRBase-referenced pre-miRNAs, 365 of them are expressed in the CNS. (B) Predictive structures of novel miRNAs were calculated with the RNAfold program. Colors represent the probability of matching between the bases. The predictive structures of novel miRNAs are similar to those of known miRNAs. (C) Histogram of the number of known and novel precursors of miRNAs expressed in each structure. (D) Venn diagramm of the repartition of pre-miRNAs in the CNS structures considering known and novel pre-miRNAs.
- Figure S1. Predictive structure of rno-miR-124-1.
Predictive structure of rno-miR-124-1 has been performed with the RNAfold program. Colors represent the probability of matching between the bases.
- Figure 2. Chromosomic repartition of miRNAs.
(A) Pie charts of the repartition of pre-miRNAs per DNA strand. There are more novel miRNAs on the positive strand than for miRBase-referenced miRNAs, Fisher exact test, P < 0.05. (B) Histogram of the repartition of known and novel pre-miRNAs along the 22 chromosomes of the rat genome. (C) Correlative analysis of the number of miRNAs in function of the length of chromosomes, R2 = 0.3814, P = 0.0022. (D) Pie chart represents genome mapping of the novel pre-miRNAs: most of the new miRNAs are intergenic (57.94%) or intronic (28.04%). Genome browser view of an intergenic pre-miRNA (novel-miR-42, E) and an intronic pre-miRNA (novel-miR-13, F).
- Figure S2. Chromosomal repartition of miRBase-referenced miRNAs expressed in the five CNS structures.
Considering rat miRNAs already referenced in miRBase, chromosomes 1, 6, and X contain the highest number of miRNAs (64, 73, and 59, respectively). Analysis of the expression of the miRBase-referenced miRNAs in the five CNS structures studied shows that chromosomes 1, 6, and X host the highest number of miRNAs (37, 61, and 45, respectively).
- Figure 3. Analysis of the sequence of novel miRNAs.
(A) The nucleotidic composition of all novel miRNAs was compiled, and the probability of each nucleotide at each position is plotted; U is the predominant nucleotide at the 5′ position. (B) Proof of concept for the “functional” ortholog miRNA hypothesis, mmu-miR-676-3p, and novel-rno-miR-21-5p share the same seed region on the LCE2D 3′UTR. Luciferase experiment proves that both miRNAs are able to interact with LCE2D 3′UTR and mediate luciferase translation inhibition. Cel-miR-67, a miRNA from C. elegans known to have no target in mammals, is used as control. Data shown are mean ± SD; numbers within the bars indicate biological replicates, one-way ANOVA, followed by the Dunnett post hoc test, ***P < 0.001, *P < 0.05.
- Figure 4. Hierarchical clustering of the miRNome of the different CNS structures.
(A, B) Heatmaps representing the expression of each miRNA from all the studied structures. miRNAs are represented at the bottom, biological replicates of the different CNS structures are represented on the right, and statistical dendrogram of clusterization of the samples is represented on the left. Colors represent the level of miRNA expression (log2 of count per million); red: high expression; green: low expression. (A) Considering miRBase-referenced miRNAs, all biological replicates of the same structure are grouped together, except cortex replicate #3. (B) Hierarchical clustering of novel miRNAs shows a perfect clustering of the biological replicates from the same structure. Black arrowheads indicate miRNAs those expression are strongly different in a structure compared with all other samples.
- Figure 5. Differentially expressed miRNAs between the structures.
All the depicted miRNAs are statistically dysregulated in the structure considered compared with all other structures as assessed with the DESeq2 algorithm and Wald test (P < 0.05). The log2 of the fold change of over- (green) and down-regulated (red) miRNAs is indicated; miRNAs written in bold correspond to novel miRNAs. (A) The olfactory bulb shows the highest number of differentially regulated miRNAs: 44 are up-regulated and 34 are down-regulated. In the cortex (B), four miRNAs are significantly up-regulated and seven are down-regulated, whereas in the hippocampus (C), only one miRNA is overexpressed and one is underexpressed. (D) In the striatum, 20 and 2 miRNAs are up- and down-regulated, respectively. (E) In the spinal cord, 32 and 34 miRNAs are up- and down-regulated, respectively.
- Figure 6. Enrichment/depletion of miRNAs in specific structures.
To focus on the miRNAs that could have the most relevant role in transcriptome regulation, we defined as enriched miRNAs those with a fold change >16, and depleted miRNAs those with a fold change <−16. Except for the striatum, all structures express specifically enriched or depleted miRNAs. The olfactory bulb shows the highest number of specific miRNAs (13 miRNAs), the spinal cord expresses 11 specific miRNAs, the cortex and the hippocampus exhibit only one specifically depleted miRNA, and interestingly, the cortex-specific miRNA is a novel miRNA (novel miRNAs are written in bold).
- Figure 7. Olfactory bulb–specific miRNAs and their regulated targets.
(A) Concerning specifically enriched miRNAs, cumulative frequency distribution of expression changes of all predicted targets is significantly different from nontarget genes (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows a down-regulation compared with nontarget genes. (B) Individual gene statistical analysis revealed that 12 target genes of the enriched miRNAs are statistically down-regulated (DESeq2 algorithm and Wald test, P < 0.05). Interestingly, some regulated genes are predicted to be the target of multiple miRNAs (gene names written in italics). Data shown are mean ± SD form three biological replicates. (C) Concerning specifically depleted miRNAs, cumulative frequency distribution of expression changes of all predicted targets is significantly different from nontarget genes (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows an up-regulation compared with nontarget genes. (D) Individual gene statistical analysis revealed that 40 target genes of miR-544-3p are up-regulated (DESeq2 algorithm and Wald test, P < 0.05); only the 20 most regulated are depicted. Data shown are mean ± SD form three biological replicates. (E) GO term enrichment analysis of the selected targets.
- Figure 8. Cortex-specific miRNA and its regulated targets.
(A) Cumulative frequency distribution of expression changes of all predicted targets is significantly shifted to the right compared with nontarget genes, revealing a global up-regulation (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows an up-regulation compared with nontarget genes. (B) Individual gene statistical analysis revealed that 20 target genes of novel-miR-28-3p are up-regulated (DESeq2 algorithm and Wald test, P < 0.05). Data shown are mean ± SD form three biological replicates. (C) GO term enrichment analysis of the selected targets.
- Figure 9. Hippocampus-specific miRNA and its regulated targets.
(A) Cumulative frequency distribution of expression changes of all predicted targets is significantly shifted to the right compared with nontarget genes, revealing a global up-regulation (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows an up-regulation compared with nontarget genes. (B) Individual gene statistical analysis revealed that 70 target genes of novel-miR-28-3p are up-regulated (DESeq2 algorithm and Wald test, P < 0.05); only the 20 most regulated are depicted. Data shown are mean ± SD form three biological replicates. (C) GO term enrichment analysis of the selected targets.
- Figure 10. Spinal cord–specific miRNAs and their regulated targets.
(A) Concerning specifically enriched miRNAs, cumulative frequency distribution of expression changes of all predicted targets is significantly different from nontarget genes (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows a down-regulation compared with nontarget genes. (B) Individual gene statistical analysis revealed that 71 target genes of the enriched miRNAs are statistically down-regulated (DESeq2 algorithm and Wald test, P < 0.05); only the 20 most regulated are depicted. Interestingly, some regulated genes are predicted to be the target of multiple miRNAs (gene names written in italics). Data shown are mean ± SD form three biological replicates. (C) Concerning specifically depleted miRNAs, cumulative frequency distribution of expression changes of all predicted targets is significantly different from nontarget genes (blue line versus black line, Kolmogorov–Smirnov test). Distribution of expression changes of statistically regulated target genes (red) shows an up-regulation compared with nontarget genes. (D) Individual gene statistical analysis revealed that 91 target genes of the depleted miRNAs are up-regulated (DESeq2 algorithm and Wald test, P < 0.05); only the 20 most regulated are depicted. Interestingly, some regulated genes are predicted to be the target of multiple miRNAs (gene names written in italics). Data shown are mean ± SD form three biological replicates. (E) GO term enrichment analysis of the selected targets.
Tables
Name of the novel miRNA Number of copies Chromosomic location Number of chromosomes rno-novel-miR-2-5p 6 2 x chr 1; chr 7; chr 12; chr 15; chr 18 5 rno-novel-miR-8-3p 3 3 x chr 1 1 rno-novel-miR-12-5p 3 chr 2; 2 x chr 4 2 rno-novel-miR-19-5p 2 chr 3; chr 20 2 rno-novel-miR-22-5p 5 chr 3; 2 x chr 5, chr 6; chr 7 4 rno-novel-miR-26-3p 2 chr 18; chr 5 2 rno-novel-miR-52-3p 2 chr 10; chr 11 2 rno-novel-miR-66-5p 2 chr 12; chr 14 2 Duplicated miRNAs are localized either on the same chromosome or on different chromosomes.
Novel rat miRNAs Mouse orthologs Human orthologs Name Sequence Name Sequence Name Sequence rno-novel-miR-7-5p AGUCAGGCUACUGGUUAUAUUCCA mmu-miR-344-5p AGUCAGGCUCCUGGCUAGAUUCCAGG N/A N/A rno-novel-miR-8-3p GGUAUAACCAAAGCCCGACUGA mmu-miR-344h-3p GGUAUAACCAAAGCCCGACUGU N/A N/A rno-novel-miR-14-3p UCUCUGGGCCUGUGUCUU mmu-miR-330-5p UCUCUGGGCCUGUGUCUUAGGC hsa-miR-330-5p UCUCUGGGCCUGUGUCUUAGGC rno-novel-miR-24-5p UCAAGAGCAAUAACGAAAA mmu-miR-335-5p UCAAGAGCAAUAACGAAAAAUGU hsa-miR-335-5p UCAAGAGCAAUAACGAAAAAUGU rno-novel-miR-35-5p UUUCCUCUCUGCCCCAUAGGGU mmu-miR-3059-5p UUUCCUCUCUGCCCCAUAGGGU N/A N/A rno-novel-miR-37-3p GCAGGAACUUGUGAGUCU mmu-miR-873a-5p GCAGGAACUUGUGAGUCUCCU hsa-miR-873-5p GCAGGAACUUGUGAGUCUCCU rno-novel-miR-38-5p ACUCUAGCUGCCAAAGGCGCU mmu-miR-1251-5p ACUCUAGCUGCCAAAGGCGCU hsa-miR-1251-5p ACUCUAGCUGCCAAAGGCGCU rno-novel-miR-56-5p ACUGGACUUGGAGUCAGAAG mmu-miR-378c ACUGGACUUGGAGUCAGAAGC hsa-miR-378a-3p ACUGGACUUGGAGUCAGAAGGC rno-novel-miR-64-5p CUAAGGCAGGCAGACUUCAGUGU mmu-miR-6540-5p CUAAGGCAGGCAGACUUCAGUG N/A N/A rno-novel-miR-72-3p CCAGUAUUGACUGUGCUGCUGAA mmu-miR-16-1-3p CCAGUAUUGACUGUGCUGCUGA hsa-miR-16-1-3p CCAGUAUUAACUGUGCUGCUGA rno-novel-miR-87-5p GUUCCUGCUGAACUGAGCCAGU mmu-miR-3074-5p GUUCCUGCUGAACUGAGCCAGU hsa-miR-3074-5p GUUCCUGCUGAACUGAGCCAG rno-novel-miR-90-5p AGGUCCUCAGUAAGUAUUUGUU mmu-miR-1264-5p AGGUCCUCAGUAAGUAUUUGUU N/A N/A Novel-miRNAs with their corresponding orthologous sequences in the mouse and/or human. We defined as ortholog an miRNA sequence with a minimum of 95% similarities with an miRNA from another species.
- Table 3.
Novel miRNAs with their corresponding functional orthologous sequences in the mouse and/or human.
Name Sequence Name Sequence Name Sequence rno-novel-miR-78-5p UGCCCCUGCUGAGGCUGUGUCU mmu-miR-3070-5p AGCCCCUGACCUUGAACCUGGGAAGCCCCUGACCUUGAACCUGGGA N/A N/A rno-novel-miR-79-3p UUAGGACUCUGGUCAUCUUUGG N/A N/A hsa-miR-3117-3p AUAGGACUCAUAUAGUGCCAGAUAGGACUCAUAUAGUGCCAG rno-novel-miR-60-5p CAGGGAGAGAACUAGUACAG N/A N/A hsa-miR-6760-5p CAGGGAGAAGGUGGAAGUGCAGACAGGGAGAAGGUGGAAGUGCAGA rno-novel-miR-86-3p ACAGACUGGUGCCUGGGUGUGG N/A N/A hsa-miR-4494 CCAGACUGUGGCUGACCAGAGGCCAGACUGUGGCUGACCAGAGG rno-novel-miR-63-5p GCCUGAGAGCUGGGGGUA N/A N/A hsa-miR-4324 CCCUGAGACCCUAACCUUAACCCUGAGACCCUAACCUUAA rno-novel-miR-21-5p CCGUCCUGUACUGCAGCA mmu-miR-676-3p CCGUCCUGAGGUUGUUGAGCUCCGUCCUGAGGUUGUUGAGCU hsa-miR-676-3p CCGUCCUGAGGUUGUUGAGCUCCGUCCUGAGGUUGUUGAGCU rno-novel-miR-54-3p UGUGUCUUUCUCCCUCUUGAC N/A N/A hsa-miR-4711-3p CGUGUCUUCUGGCUUGAUCGUGUCUUCUGGCUUGAU rno-novel-miR-57-3p CUCUCACCUCCCGCUCUCCACA N/A N/A hsa-miR-1229-3p CUCUCACCACUGCCCUCCCACAGCUCUCACCACUGCCCUCCCACAG rno-novel-miR-48-5p AUGGCGGCUGUGAGUCUG N/A N/A hsa-miR-6770-3p CUGGCGGCUGUGUCUUCACAGCUGGCGGCUGUGUCUUCACAG rno-novel-miR-84-5p CUGUGGGCUGCAGGAGGAC mmu-miR-134-3p CUGUGGGCCACCUAGUCACCCUGUGGGCCACCUAGUCACC hsa-miR-134-3p CUGUGGGCCACCUAGUCACCCUGUGGGCCACCUAGUCACC rno-novel-miR-80-3p CCCAGGGAGCUGUAAGAGCCG mmu-miR-7226-5p GCCAGGGAAGUUGAUUGUGUGAAGGGGCCAGGGAAGUUGAUUGUGUGAAGGG N/A N/A rno-novel-miR-59-3p UCCCCUGGGUCUGUGCUCUGCA mmu-miR-331-3p GCCCCUGGGCCUAUCCUAGAAGCCCCUGGGCCUAUCCUAGAA hsa-miR-331-3p GCCCCUGGGCCUAUCCUAGAAGCCCCUGGGCCUAUCCUAGAA rno-novel-miR-25-3p GCGGGCGGGCGGGAGGCG mmu-miR-5126 GCGGGCGGGGCCGGGGGCGGGGGCGGGCGGGGCCGGGGGCGGGG N/A N/A rno-novel-miR-3-3p AUAAGUGUAGAGAGUCUGUAGU mmu-miR-668-5p GUAAGUGUGCCUCGGGUGAGCAUGGUAAGUGUGCCUCGGGUGAGCAUG N/A N/A rno-novel-miR-71-5p CUCCCUCUAGUCCUCUUGGUUGU N/A N/A hsa-miR-642a-5p GUCCCUCUCCAAAUGUGUCUUGGUCCCUCUCCAAAUGUGUCUUG rno-novel-miR-9-3p CUGGGCGGGAUGGGAGGUGG N/A N/A hsa-miR-1228-5p GUGGGCGGGGGCAGGUGUGUG rno-novel-miR-17-5p GCAAGGCCCAGCGAGUGACU N/A N/A hsa-miR-3922-5p UCAAGGCCAGAGGUCCCACAGCA rno-novel-miR-41-5p UCCGGGGCUGCGGGAUGA mmu-miR-673-3p UCCGGGGCUGAGUUCUGUGCACC N/A N/A rno-novel-miR-32-5p ACUCGGAUCAAGCUGAGAGCCA mmu-miR-6336 UCUCGGAUUUAGUAAGAGAUC N/A N/A rno-novel-miR-49-5p ACUCUCUCACUCUGCAUGGUUA N/A N/A hsa-miR-7110-3p UCUCUCUCCCACUUCCCUGCAG rno-novel-miR-73-3p UGGAGGAGAGAAAAAGAGA N/A N/A hsa-miR-765 UGGAGGAGAAGGAAGGUGAUG rno-novel-miR-77-5p AGUUUUCUGCCUUUCGCUCUGUGG mmu-miR-7214-5p UGUUUUCUGGGUUGGAAUGAGAA N/A N/A rno-novel-miR-10-3p AUUCUUCUCAGUGGGCUUAGA mmu-miR-6946-3p UUUCUUCUCUUCCCUUUCAG N/A N/A Functional orthologous sequences were determined with a perfect homology into the seed region (italicized sequence).
Structure Specific miRNA Regulated target mRNA mRNA function associated with the structure Olfactory bulb miR-544-3p Sp8 Transcription factor known to regulate olfactory bulb interneuron development (Li et al, 2018) Barhl2 Transcription factor involved in the development of region-specific differences in the forebrain (Parish et al, 2016) and the diencephalon (Ding et al, 2017) Dcx Mandatory for proper migration and development of olfactory bulb neurons (Belvindrah et al, 2011) Wnt5a Necessary for olfactory axon connections (Zaghetto et al, 2007; Paina et al, 2011; Pino et al, 2011) Cortex novel-miR-28-3p Vip Vip-expressing interneurons are crucial for cortical circuits development (Batista-Brito et al, 2017) Cbln2 Expressed in the subpopulation of excitatory cortical neurons (Seigneur & Südhof, 2017) Lynx1 Mandatory for cortical network stability (Morishita et al, 2010) Hippocampus miR-3065-5p Lhx9 Implicated in the development of the hippocampal subdivisions (Abellán et al, 2014) Il16 Modulation of Kv4.2K+ currents and thus neuronal intrinsic properties (Fenster et al, 2007) Gnrhr Involved in the hippocampus-specific neuronal plasticity mechanism (Schang et al, 2011) Prkcg Supports hippocampal long-term potentiation (Gärtner et al, 2006) Pcdh19 Regulated in plasticity paradigm (Kim et al, 2010) and mutations linked to epilepsy and mental retardation (Dibbens et al, 2008) Gria1 Involved in neuronal homeostatic plasticity (Sutton et al, 2006; Letellier et al, 2014) Spinal Cord miR-10b-5p Bcl11b Mandatory for the development of axonal projections (Arlotta et al, 2005) miR-615 Slc17a7 Defines a subpopulation of neurons involved in pain processing (Landry et al, 2004; Brumovsky et al, 2007) FosB Increased expression during spinal cord development and nociception (Herdegen et al, 1991; Redemann-Fibi et al, 1991) Arc Enhanced expression in response to pain (Hossaini et al, 2010) Synpo Regulated in bone cancer pain conditions (Elramah et al, 2017) miR-344g Pax2 Involved in spinal cord development (Larsson, 2017) Lmx1b Involved in spinal cord development (Ding et al, 2004; Hilinski et al, 2016) Lbx1 Involved in pain mechanisms (Gross et al, 2002; Cheng et al, 2017) Aplnr Involved in pain mechanisms (Xiong et al, 2017) miR-551b-5p Hoxb3 Involved in spinal cord development (Yau et al, 2002) Pax8 Involved in spinal cord development (Batista & Lewis, 2008) Nkx6-1 Involved in spinal cord development (Sander et al, 2000; Vallstedt et al, 2001) Glp1r Involved in pain mechanisms (Djenoune et al, 2014) Pkd2l1 Defines a subpopulation of neurons regulating locomotion (Böhm et al, 2016) For each structure, specifically enriched or depleted miRNAs (in bold or italic, respectively) were associated with oppositely regulated mRNAs (up-regulated in bold and down-regulated in italic) known for their role in the given CNS structure.
Table S6 miRNA cluster analysis.
