Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs

Abstract

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤ 0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

Keywords: RNA sequencing; isomIRs; microRNAs; noncoding RNA; transcriptome.

Fig. 1.

Flow diagram depicting the steps taken in identifying novel miRNAs. Shown is a flow diagram of the process to identify candidate novel miRNAs from 1,323 deep-sequencing samples using miRDeep2. Only mature miRNA with associated FDR ≤ 0.05 were kept for further analysis. Discovered sequences that were present in release 20 of miRBase, or overlapped known tRNAs, snRNAs, or rRNAs were discarded. A total of 3,707 candidate miRNAs derived from 3,494 precursor sequences were identified. Intersection of the identified miRNAs with 43 Ago-CLIP-seq samples showed evidence of Ago loading for 1,657 newly discovered miRNAs. Sixty-six of the identified precursors produced two miRNAs, one from each arm: one product was supported by Ago CLIP-seq whereas the other was not.

Fig. 2.

Both known and novel miRNAs are encoded throughout the genome. Shown are the regions of the genome from which miRNAs of miRBase (A) and the novel miRNAs (B) are encoded. All annotations for genes [3′ UTR, coding DNA sequence (CDS), and 5′ UTR], long noncoding RNAs (lncRNAs), and pseudogenes are from release 72 of ENSEMBL; all repeat regions are from RepeatMasker. Intronic regions are defined to be those segments of known unspliced pre-mRNA that remain after removing all known genomic features that are sense to the pre-mRNA such as exons, miRNAs, repeat elements, etc. Intergenic regions are defined to be those segments of the genome that remain after removing all protein coding loci as well as all other already-characterized genomic features.

Fig. 3.

Novel miRNAs display a tissue-specific pattern of expression. Shown are the Jaccard index value for the overlap of expressed miRNAs between any two tissues for the novel miRNAs (A) and the miRBase miRNAs (B). A miRNA was considered to be expressed in each tissue if the miRNA had a normalized expression of ≥1/100 the expression of endogenous SNORD44. (C) Principal-component analysis of the sequence data can cluster the samples based upon tissue types.

Fig. 4.

Dicer knockdown results in a decrease in miRNA expression. Fold change in miRNA expression levels in MCF7 cells after Dicer knockdown for release 20 miRBase miRNAs (blue), all newly discovered miRNAs (red), and the subset of Ago CLIP-seq–supported newly discovered miRNAs (green). y axis, percentage of expressed miRNAs; x axis, fold change in expression of Control vs. Dicer knockdown. A negative fold change equals decrease of the miRNA in the knockdown. inf, miRNA was absent in either the Dicer knockdown (−inf) or the Control sample (inf).

Fig. 5.

Expression of novel miRNAs in a variety of cell lines and tissue types. Stem-loop RT-PCR experiments for 20 newly discovered miRNAs (one miRNA per row). Each row represents a specific cell line.

Fig. 6.

Examples of novel mature miRNAs from previously uncharacterized arms of precursors linked to important cell processes. (A) Novel miRNA TJU_CMC.MD2.ID00400.5p-miR arises from the 5′ arm of miR-107’s precursor (MI0000114; chr10:91,352,549-91,352,572). (B) Novel miRNA TJU_CMC.MD2.ID02736.5p-miR arises from the 5′ end of the miR-103-a- precursor (MI0000109; chr5:2167,987,901-167,987,978). The y axis is logarithmic (base 2).

Fig. 7.

Multiple sequence alignments of seed-based paralogues. The alignments shown in each panel comprise novel miRNAs and miRBase miRNAs that have been clustered based on their shared seed sequences (red highlight). (A–C) Novel miRNAs that are previously uncharacterized seed-paralogues of known miRNAs. (D) A new seed family consisting of 14 newly discovered miRNAs.