Since their original description in the peer-reviewed literature, microRNAs have been increasingly recognized as workhorses of gene-expression regulation. Though maybe "work ponys" is the more appropriate phrase, since miRNAs are only 20-22 nucleotides in length.

Given that shortness, it comes as no great surprise that microRNAs (miRNAs) do not code for proteins themselves. Instead, they bind to messenger RNAs and repress their translation. Unlike their cousin - silencing RNA, or siRNAs - miRNAs bind imperfectly, with some base pair mismatches. For that reason, the bound mRNA is not destroyed (as it is when bound by siRNAs). Instead, the efficiency of translating it into proteins is reduced through mechanisms that are still unknown.

Though developmental regulation, fat metabolism and gene silencing are among the areas in which miRNAs in general are thought to play a role, the biological functions of specific mammalian miRNAs have remained a matter of speculation to date. Now, two papers have been published that ascribe functional roles to two different mammalian miRNAs.

MiRNAs Have Potential In Diabetes, Obesity

In the Nov. 11, 2004, issue of Nature, researchers from New York's Rockefeller University, Lund University in Sweden, New York University and Oxford University in the UK report the identification of an miRNA that regulates insulin secretion. And in the Oct. 25, 2004, issue of the Journal of Biological Chemistry, researchers from Carlsbad, Calif.-based Isis Pharmaceuticals report on a miRNA involved in the control of fat cell differentiation.

In the Nature paper, titled "A pancreatic islet-specific microRNA regulates insulin secretion," the researchers first screened cell lines for pancreatic islet-specific miRNAs. Two of them, miR-375 and miR-376, were selected for further study on the basis of their abundance in and specificity to pancreatic islet cells. Of the two, only miR-375 had an effect on insulin secretion; insulin secretion was decreased when miR-375 was increased, and increased when miR-375 was silenced. Control experiments localized miR-375's effects to the end of the insulin secretion process, namely exocytosis, rather than changes in metabolism or signaling.

The researchers next wanted to determine the gene targets of miR-375. A bioinformatics approach yielded more than 60 potential gene targets, of which five genes were chosen for further studies.

"We concentrated on the genes we could implicate in exocytosis," said senior author Markus Stoffel, Robert and Harriet Heilbrunn professor and director of the Laboratory of Metabolic Diseases at Rockefeller University. Among the five genes they chose, two panned out: Mtpn and VTi1a. Neither had been studied in pancreatic b-cells before.

"There was not a single paper," Stoffel told BioWorld Today. However, both were known to be involved in neuronal vesicle transport and neurotransmitter release, "which, if you think about it, is a similar process."

The authors used a mix of techniques to demonstrate that miR-375 works by affecting those genes, and again, that silencing those genes inhibits insulin secretion. Though VTi1a is more closely tied to exocytosis in the scientific literature than is Mtpn, the results in the Nature paper implicated Mtpn more strongly than VTi1a. Stoffel said that might be due to experimental constraints rather than a true reflection of their relative importance, since VTi1a protein is harder to knock down and the researchers were unable to silence it completely.

Stoffel's group now is working on creating animal models with dysregulated miRNAs, to find out whether such dysregulation leads to disease and to determine whether manipulating miRNA, by either upregulating or inhibiting it, will in turn be an effective intervention.

The second paper is titled "MicroRNA-143 regulates adipocyte differentiation."

"Adipocytes are important targets in obesity, as well as Type II diabetes," said Frank Bennett, vice president of antisense research at Isis and co-author of the Journal of Biological Chemistry paper. "By blocking adipocyte differentiation, you should in theory be able to affect weight and weight accumulation." Fat cells also are a tissue type that lends itself well to oligonucleotide-based intervention, Isis' methodological focus.

In their paper, Isis scientists used a combination of expression data and functional inhibition to identify miRNAs involved in adipocyte transformation. For the functional inhibition data, cultured pre-adipocytes were transfected with antisense-oligonucleotides targeting known miRNA sequences. For functional expression data, the scientists used a microarray approach to identify miRNAs that are expressed in pre-adipocytes.

In combination, the studies pointed to a number of miRNAs as having possible roles in adipocyte differentiation. MiR-143 was chosen for validation.

"We looked at five endpoints, and miR-143 clearly affected all five," Bennett explained, adding that other miRNAs also were identified through the screen and their characterization is ongoing.

The researchers investigated whether inhibiting miR-143 would affect the genes it was thought to be inhibiting and consequently, fat cell differentiation. They chose ERK-5 for their investigations, which is a predicted target of miR-143.

When miR-143 was inhibited through antisense oligonucleotides in cultured pre-adipocytes, ERK-5 protein was upregulated and fewer differentiating cells were observed. The effect was specific; another miRNA that Isis researchers found to inhibit differentiation in the same study did not affect ERK.

A Coincidence Is Not A Trend

Though the first two papers ascribing functions to specific miRNAs both report on the general area of metabolism, their authors warn against reading significance into that fact.

"Metabolism is so essential for all cells, it is logical that people are looking there" for miRNA functions, Stoffel said, adding that a variety of other roles have been described for miRNAs in lower animals and that he believes that the same will hold true in mammals.

Bennett agreed, pointing out that miR-143 expression also is known to be decreased in a variety of human cancer cell lines, though he cautioned that the functional significance of those observations have not been investigated to date. He said he believes that "microRNAs will be found to be as broadly involved as cell transcription factors. In fact, they may be the new transcription factors for the new century, if you will."