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UTSMC Team Develops Method for Genome-Wide Primary microRNA Annotation

NEW YORK (GenomeWeb) – In an effort to better understand the mechanisms that regulate microRNA expression, a team led by researchers from the University of Texas Southwestern Medical Center (UTSMC) this week reported on a novel experimental and computational pipeline that enables the genome-wide detection and mapping of primary miRNA (pri-miRNA) transcripts.

Applying the approach to a panel of human and mouse cell lines of various origins, the investigators successfully annotated roughly 70 percent of the pri-miRNAs in these species. They also expect that the near-complete assembly of annotated miRNAs is possible through examination of other cell types.

MiRNAs are regulatory RNA molecules that are transcribed by RNA polymerase II as long pri-miRNAs that are cleaved by the RNase III enzyme Drosha into miRNA precursors, which in turn are exported into the cytoplasm where they are processed into their final form.

"One remarkable feature of primary miRNAs is their extreme length, even in cases where they function only to produce a single [approximately] 22 nucleotide miRNA," UTSWMC's Joshua Mendell, a co-author of the study, said in a statement. "Although it seems wasteful to produce such long RNAs, most of which will be immediately degraded, this organization may have arisen to allow complex mechanisms of regulation of the encoded miRNA."

It is well-established that miRNAs play a key role in regulating gene expression that control important biological processes and are frequently dysregulated in disease. "Indeed, key transcription factors and signaling pathways have been shown to strongly regulate miRNA expression under diverse physiologic and pathophysiologic conditions," the UTSMC team wrote in Genome Research. "Nevertheless, a major bottleneck in the dissection of the mechanisms through which these pathways control miRNA levels has been our incomplete understanding of miRNA gene structures."

Techniques exist for identifying the genomic locations of the promoters and transcription start sites of miRNAs, but these do not provide annotation of splicing patterns of miRNA primary transcripts and therefore fail to offer a complete picture of miRNA gene structure.

And while high-throughout RNA sequencing has become a powerful tool for transcriptome reconstruction, the low abundance of pri-miRNAs makes them poorly represented in standard RNA-seq datasets, hindering comprehensive annotation of their structures.

To overcome these issues, the UTSMC group came up with a strategy to enable genome-wide pri-miRNA reconstruction.

First, a dominant negative Drosha protein that globally impairs pri-miRNA processing is expressed in cells in order to stabilize pri-miRNA transcripts and improve their coverage in RNA-seq libraries. Then StringTie, a transcriptome assembler capable of accurately reconstructing pri-miRNAs, is applied.

Using the approach in different cell lines, the scientists were able to obtain pri-miRNA structures for a number of human and mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. They also discovered a number of human intergenic conserved miRNA clusters with alternative promoters.

"We were surprised to find cases where such miRNAs could be alternatively co-transcribed or separately transcribed, depending on which promoter is used," Mendell said.

The team further classified the pri-miRNAs into three main categories based on gene structure. The first, Class I, is transcribed independently of other genes and likely represent independent transcription units. Class II, meantime, is transcribed as an extension of a protein-coding gene, while Class III is transcribed as an extension of a noncoding RNA.

"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," the investigators concluded in their paper. "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."