Genome-wide annotation of microRNA primary transcript structures reveals novel regulatory mechanisms

Abstract

Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.

An integrated expression atlas of miRNAs and their promoters in human and mouse

“…The correlation was substantially higher for highly differentially expressed miRNAs (average Spearman r = 0.59; n = 159; P = 1 × 10 −35 , Mann-Whitney U test, one-sided), and exceeded correlations found for previously published 14,33–35 miRNA promoter annotations (Figure S20d). About 11% of pri-miRNAs in human were polycistronic, containing multiple mature miRNAs with highly correlated expression levels (average Spearman r = 0.74; n = 1,372; P < 10 −100 , Mann-Whitney U test, one-sided) (Figure 3d and S28).…”

“…RACE experiments confirmed that the transcripts generated at the identified promoter extended to the mature miRNA for 6 out of 7 miRNAs (Figure S21, Table S18). RNA-seq data 14 of cells expressing a dominant-negative Drosha protein provided additional evidence for the FANTOM5 annotated pri-miRNAs, with 483 out of 607 pri-miRNAs (80%) having a 5′ end within 300 base pairs of an RNA-seq transcript assembly extending to the mature miRNA locus (Figure S22). …”

“…Manual curation furthermore identified the promoter for an additional 196, mostly intergenic, pre-miRNAs, thereby generating the—to our knowledge—largest miRNA promoter collection to date (Table S17, Figure S20a). Across the human robust set, an associated ENCODE RAMPAGE (RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression 32 ) 5′ end was found within 300 base pairs of more than 75% of the FANTOM5 curated promoters both for intergenic and intronic miRNAs, outperforming the miRGen 33 , Chang et al 14 , miRStart 34 , and TSmiR 35 collections of miRNA promoter annotations (Figure S20b). The median distance between the FANTOM5 annotated miRNA promoter and the associated RAMPAGE 5′ end was 1 nucleotide, and was thereby closer than any of the existing miRNA promoter annotations (Figure S20c).…”

“…Profiling pri-miRNAs, which is more challenging owing to their transient character, has been accomplished by RNA-seq in cells expressing dominant-negative Drosha 14 . Additionally, since most pri-miRNAs are produced by RNA polymerase II and therefore have a 5′ cap 11 , they are amenable to Cap Analysis Gene Expression (CAGE) profiling 15,16 , which identifies the pri-miRNA transcription start site and therefore the promoter region, while directly quantifying the pri-miRNA expression level.…”

Novel determinants of mammalian primary microRNA processing revealed by systematic evaluation of hairpin-containing transcripts and human genetic variation

“…HepG2) published by the Mendell laboratory (Chang et al 2015), as well as publicly available mature miRNA sequencing data from other laboratories (Bogerd et al 2014;Cao et al 2016;Hannafon et al 2016). By examining miRNAs with a higher mature-to-primiRNA ratio versus those with a lower ratio, we observed significant enrichment for optimal stem length or CNNC, and a stronger enrichment when both features were present (Supplemental Fig.…”