Novel MicroRNAs in the Aging Mouse Brain Figure 1. Novel miRNA candidates. (a) Novel miRNA candidates that change more than 2.0-fold in expression in old versus young mouse brains. MiRNA frequency was normalized by all reads that matched to the mouse genome (mm9) (Old/Young = 1.472107). {: candidates validated by qRT- PCR. {: candidates with sequence overlap with known miRNAs (but have distinct mature miRNA sequences: isomiRs). A: Novel miRNA candidates that map to regions overlapping snoRNA and rRNA sequences (see main text). Blue font: miRNA novel to mouse. Black font: completely novel miRNA sequence, excluding seed sequence matches. Differentially expressed miRNAs with P-values ,0.05 (calculated using DEGseq [20]) indicated in bold. See also Table S4. (b) Secondary structures of putative precursor hairpins corresponding to nine novel miRNA candidates identified in this study. The predicted miRNA mature sequences are highlighted in red. Four of these novel miRNAs were found to be up-regulated (top) in aged mouse brain while five others were down-regulated (bottom). (See also Table S4). (c) Comparison of qRT-PCR data with deep sequencing data for the nine novel miRNA candidates shown in (b). Values shown are log2 ratios of old versus young brain expression levels. qPCR results were normalized to U6 snRNA expression levels. Inset: Correlation of expression changes as measured by deep sequencing versus qPCR (Pearson correlation coefficient = 0.78). Plot for miR-5620 (isomiR) was taken out as the sequence was not reliably detected by qPCR. (d) Sequence alignment of novel miRNA candidates with known miRNAs of other species. *: conserved nucleotide. age: Ateles geoffroyi. bta: Bos taurus. dan: Drosophila ananassae. der: Drosophila erecta. dgr: Drosophila grimshawi. dme: Drosophila melanogaster. dmo: Drosophila mojavensis. dpe: Drosophila persimilis. dps: Drosophila pseudoobscura. dse: Drosophila sechellia. dsi: Drosophila simulans. dvi: Drosophila virilis. dwi: Drosophila willistoni. dya: Drosophila yakuba. gga: Gallus gallus. ggo: Gorilla gorilla. hsa: Homo sapiens. lla: Lagothrix lagotricha. mdo: Monodelphis domestica. mml: Macaca mulatta. mne: Macaca nemestrina. ppa: Pan paniscus. ppy: Pongo pygmaeus. ptr: Pan troglodytes. rno: Rattus norvegicus. sla: Saguinus labiatus. sme: Schmidtea mediterranea. xtr: Xenopus tropicalis. doi:10.1371/journal.pone.0040028.g001 Figure 2. Genomic locations of brain-expressed miRNAs found within 10 kb region of each other. (a) miRNA clusters found on chromosome 12. Top panel: location of miRNAs relative to the entire chromosome 12. Bottom three panels: three distinct clusters found in the boxed off region in the top panel. The three clusters are located close to each other. (b) miRNA clusters found on X chromosome. Top panel: location of miRNAs relative to the entire X chromosome. Bottom two panels: clusters found in the boxed off regions of the top panel, from left to right. Blue: brain-expressed known miRNAs. Teal: miRNAs downregulated in expression in our dataset. Purple: miRNAs upregulated in expression in our dataset. Pink: known miRNAs found in the same genomic region but not found to be expressed in our dataset. Red: brain-expressed novel miRNA candidates. Green: TROMER transcriptome data (retrieved from UCSC Genome Browser). doi:10.1371/journal.pone.0040028.g002 PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40028