A new cell-based, genome-wide study has identified a cluster of micro RNAs (miRNAs)—noncoding RNA molecules about 22 nucleotides long—that constitute a new layer of modulation for clock genes responsible for circadian rhythms. Until now, most studies probing regulatory mechanisms of daily biological rhythms have focused on feedback loops that regulate protein-coding genes.

Regulation of circadian rhythms is key, not only in understanding sleep, but also diseases such as Alzheimer’s, cancer, and diabetes. Understanding how miRNAs regulate the circadian clock in different tissues and organs could reveal new therapeutic approaches.

These findings are reported in the article titled, “A genome-wide microRNA screen identifies the microRNA-183/96/182 cluster as a modulator of circadian rhythms,” published in the Proceedings of the National Academy of Sciences.

“We’ve seen how the function of these clock genes are really important in many different diseases,” said Steve Kay, PhD, DSc, provost professor of neurology, biomedical engineering, and quantitative computational biology at the Keck School of Medicine of the University of Southern California. “But what we were blind to was a whole different funky kind of gene network that also is important for circadian regulation and this is the whole crazy world of what we call noncoding microRNA.”

Kay headshot
Steve Kay, PhD, DSc, provost professor of neurology, biomedical engineering, and quantitative computational biology at the Keck School of Medicine of the University of Southern California.

Previously mislabeled “junk DNA,” miRNAs are now known to affect gene expression by silencing the post-transcriptional machinery that generates proteins from messenger RNAs. That miRNAs may also regulate circadian clocks had been revealed in earlier studies but the identity of the miRNAs that actually do so remained unknown.

In collaboration with the Genomics Institute of the Novartis Research Foundation (GNF) in San Diego, with automation-enabled high-throughput experimentation capacity, Kay and his team, led by Lili Zhou, PhD, research associate in the Keck School’s department of neurology, developed a high throughput automated screen individually transferring miRNAs into luciferase reporter cells engineered to glow on and off, based on the cell’s 24-hour circadian clock cycle.

“The collaboration with GNF made it possible for us to conduct the first cell-based, genome-wide screening approach to systematically identify which of the hundreds of miRNAs might be the ones modulating circadian rhythms,” said Zhou.

The authors screened a library of 989 miRNAs and found 120 miRNAs changed the length of the circadian period in U2OS osteosarcoma reporter cells.

“Much to our surprise,” said Kay, “we discovered about 110 to 120 miRNAs that do this.”

Of the 120 miRNAs, the authors focused on the circadian regulatory function of a specific miRNA cluster, miR-183/96/182, validating the results both in vitro and in vivo. They validated that all three members of this miRNA cluster modulate circadian rhythms and miR-96 regulates a core circadian clock gene, PER2.

Kay’s team including Caitlyn Miller, a biochemistry undergraduate from the University of Southern California, Dornsife, then validated the findings by inactivating each miRNA to gauge its effect on cellular circadian rhythms.

Knocking out the miRNAs had the opposite effect on the cells’ circadian rhythm as adding them to the cells. Knocking out miR-183 or miR-182 shortened the circadian period length while knocking out  miR-96 or miR-182 increased the amplitude, revealing results that were opposite from the observed effects when the miRNAs were added into the cells.

The authors also investigated the physiologic and behavioral effects of miRNAs in mice. They knocked out miR-183/96/182 in mice and saw that inactivating the cluster interfered with the ability of mice to run on a wheel in the dark. They then examined the effect of inactivating the miRNA cluster in brain, retina, and lung tissue, and revealed the miRNAs regulate the circadian clock is tissue specific.

“In the brain we’re interested in connecting the clock to diseases like Alzheimer’s, in the lung we’re interested in connecting the clock to diseases like asthma,” said Kay. “The next step I think is for us to model disease states in animals and in cells and look at how these microRNAs are functioning in those disease states.”

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