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 stemloop 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. SignificanceMicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology.

The mammalian genome is transcribed in a developmentally regulated manner, generating RNA strands ranging from long to short non-coding RNA (ncRNAs). NcRNAs generated by intergenic sequences and protein-coding loci, represent up to 98 % of the human transcriptome. Non-coding transcripts comprise short ncRNAs such as microRNAs, piwi-interacting RNAs, small nucleolar RNAs and long intergenic RNAs, most of which exercise a strictly controlled negative regulation of expression of protein-coding genes. In humans, the DLK1-DIO3 genomic region, located on human chromosome 14 (14q32) contains the paternally expressed imprinted genes DLK1, RTL1, and DIO3 and the maternally expressed imprinted genes MEG3 (Gtl2), MEG8 (RIAN), and antisense RTL1 (asRTL1). This region hosts, in addition to two long intergenic RNAs, the MEG3 and MEG8, one of the largest microRNA clusters in the genome, with 53 miRNAs in the forward strand and one (mir-1247) in the reverse strand. Many of these miRNAs are differentially expressed in several pathologic processes and various cancers. A better understanding of the pathophysiologic importance of the DLK1-DIO3 domain-containing microRNA cluster may contribute to innovative therapeutic strategies in a range of diseases. Here we present an in-depth review of this vital genomic region, and examine the role the microRNAs of this region may play in controlling tissue homeostasis and in the pathogenesis of some human diseases, mostly cancer, when aberrantly expressed. The potential clinical implications of this data are also discussed.