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Stanford Team Reports Novel Regulatory Role for microRNA Precursors

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NEW YORK (GenomeWeb) – Researchers from Stanford University this week reported new data showing that microRNA precursors, long thought to only be intermediates in the miRNA biogenesis pathway, are capable of competing with mature miRNAs, impeding their ability to repress target genes.

The findings reveal such precursors as a new class of post-transcriptional miRNA regulators, and may point to new approaches for using miRNAs therapeutically, according to the investigators.

To date, miRNAs have been extensively studied, and the means by which they are generated is well understood. After transcription, long primary miRNA (pri-miRNA) transcripts are processed to 60- to 80-nucleotide long precursor miRNAs (pre-miRNAs), which are subsequently cleaved into mature miRNAs roughly 22 nucleotides in length.

But in the case of one miRNA — miR-151 — this system is not so straightforward. In 2007, a team from the Wistar Institute reported on the discovery that pre-miR-151 is A-to-I edited. This inhibits additional processing by Dicer, leading to the accumulation of the edited precursor and reduced mature miR-151 levels in the mammalian brain.

"However, the fate of the edited miR-151 precursor is not known, nor is the importance of miR-151 editing in brain well appreciated," the Stanford team, which was led by Mark Kay, wrote in a paper appearing in Nature Structural & Molecular Biology.

For its part, miR-151 arises from two head-to-head L2c LINE integration events in intron 21 of the Ptk2 gene that lead to expression of a hairpin that is targeted by Drosha and Dicer and cut into a bona fide miRNA, the researchers explained.

However, a high degree of complementarity to miR-151 can be found in the 3' UTRs of several genes in which L2 LINE integration and duplication events have occurred. For instance, the 3' UTR of human gene E2F6 contains a perfect match for the guide strand of mature miR-151, or miR-151-5p. Meantime, a single mismatch is present in the seed region of miR-151-5 in the '3 UTR of the mouse form of E2F6.

E2F6 is a protein within the E2F family of transcription factors that helps regulate cell cycles.

Given the single mismatch in the mouse E2F6-miR-151 pair, Kay's team set out to confirm whether miR-151 can silence the gene and, if so, identify the mechanism responsible for its doing so.

In their experiments, the researchers ultimately demonstrated mouse pri- or pre-miR-151 can bind to and compete with both mature miR-151-5p and its passenger strand, or miR-151-3p, for binding sites contained within the complementary regions of the mRNA 3' UTR of murine E2F6.

Mouse E2F6 mRNA levels, they showed, were directly regulated by both the pri and pre-miRNA. Additionally, miR-151-mediated repression of ARHGDIA — an established miR-151-5p target that lacks extensive complementarity with the miRNA's guide strand — was found to be dependent on the level of mature miR-151 because only the mature miRNA binds the 3' UTR.

Taken together, these findings suggest that miR-151 processing can have different effects on separate mRNA targets within a cell, the team wrote in Nature Structural & Molecular Biology.

Since the ratio of miRNA precursors to mature forms varies for many miRNAs both during development and in cancer, the Stanford group thought that there might be other miRNA precursors with direct regulatory roles in target gene expression. To find out, they developed a bioinformatics pipeline combining miRNA target site predictions with RNA folding analyses.

They discovered 138 miRNAs predicted to target 1,607 mRNAs common to mice and humans, and for which pre-miRNAs were predicted to compete with their corresponding mature miRNAs for targets. They selected one for further study: miR-124, which targets SNAI2, a transcription factor overexpressed in a number of cancers.

"As expected, the presence of pre-miR-124 and mature miR-124 resulted in lesser repression of the SNAI2 target than did mature miR-124 only," they wrote in their paper. "This suggested that pre-miR-124 limited the accessibility of mature miR-124 to its [miRNA-response elements] in the SNAI2 3' UTR."

Combined with the bioinformatics results, these data indicate that this type of miRNA-mediated gene silencing "might be applicable to a substantial number of endogenous miRNAs," they added. Indeed, "it is likely that precursor miRNAs competing with mature miRNAs and affecting their activity constitute a common regulatory event" — one that might be critical in development and cancer.

"Our results challenge the dogma in which miRNA precursors are considered to be mere nonfunctional intermediates in the miRNA biogenesis pathway," they concluded.

Given the Kay lab's interest in gene therapy and small RNA-based gene regulation, the researchers see the findings as also having implications for cancer research, Biswajoy Roy-Chaudhuri, lead author of the Nature Structural & Molecular Biology paper, told Gene Silencing News.

"In certain cancers … there is often an accumulation of … intermediates in the [miRNA] biogenesis pathway," he explained. To date, however, most research into the role of miRNAs in the disease have focused on the mature forms of these non-coding RNAs. This newest study suggests that pri- and pre-miRNAs are worth a look, as well.

"When there is a decline in tumor suppressor microRNAs, then often there is a rise in the oncogenic target," Roy-Chaudhuri added. "Our model says when you have a decline in the natural form of the microRNAs, the precursor accumulation could also have an extra [impact] on the regulation by affecting the activity of this already reduced population of the natural microRNA."

Further, the discovery of the effect of miRNA precursors on gene silencing could help in the creation of targeted miRNA therapeutics, Paul Valdmanis, second author on the study, noted. Because miRNAs can repress expression of multiple gene targets, for instance, it may be possible to deliberately design a pre-miRNA so that it interferes with miRNA repression of one gene while leaving the repression of others unaffected, he told Gene Silencing News.

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