(SALT LAKE CITY)—Dicer, an enzyme essential for life, cleaves long double-stranded RNA (dsRNA) into short pieces that regulate gene expression. But in many ways dsRNA molecules all look alike, and Dicer has to make some important decisions: Is this a viral dsRNA that needs to be cleaved to trigger an immune response, or a friendly dsRNA that needs to be cleaved to make my own cells work properly?
In a study using Dicer from Drosophila (fruit flies), published online today in Molecular Cell, University of Utah researchers show that Dicer uses unique features at the ends of dsRNA molecules, much the way we use human faces, to tell the difference between friend or foe.
When Dicer encounters dsRNA whose two strands end at the same place, called "blunt" dsRNA, it clamps around the dsRNA so that it can efficiently cleave the molecule along its entire length to make many small interfering RNAs, or siRNAs. While further studies are necessary to provide proof, the authors speculate this type of "processive" reaction may be uniquely suited for cleaving viral dsRNA to trigger an antiviral response.
When two strands of dsRNA do not end at the same place, and one strand "overhangs," Dicer reacts differently. The authors show that when Dicer encounters a specific type of overhang, called a 3' overhang, it does not clamp, but falls off of the dsRNA after every cleavage. This type of cleavage is required to make small RNAs, which are important for the normal function of cells.
"Understanding that Dicer can tell the difference between blunt and 3' overhangs is likely the tip of the iceberg," says Brenda L. Bass, Ph.D., distinguished professor, H.A. and Edna Benning Presidential Endowed Chair in Biochemistry and the study's corresponding author. In fact, as part of the study, doctoral student and co-author Kyle Trettin showed that an accessory protein called Loquacious-PD can allow Dicer to recognize ends that it normally can't see.
Dicer is a very big protein, with lots of moving parts. One of its parts is called a helicase domain, and the authors show that this domain is required for Dicer to change its shape when it clamps onto blunt dsRNA. Big proteins are often hard to overexpress, or produce in large amounts, making it difficult to do biochemistry studies such as the one performed by Bass' laboratory.
In fact, problems in overexpressing Dicer have hindered progress in understanding how Dicer works. A key aspect of the new study are the protein purification protocols developed by doctoral student and first author Niladri K. Sinha, who spent 18 months carefully optimizing every step of the overexpression and protein purification. Bass speculates that Sinha now has more Dicer than anyone else in the world, but notes they have included careful protocols in their publication so as to allow any researcher to obtain large quantities of Dicer.
Understanding how Dicer discriminates between viral dsRNA and the normal dsRNA made in our cells may allow us to understand diseases that result when the process goes awry. "For example, other studies in our lab are addressing the hypothesis that aberrant immune responses, such as the inflammation that accompanies diabetes and other diseases, may occur when a cell's normal dsRNA is misrecognized as a virus," Bass says.
This study was funded by the H.A. and Edna Benning Society at the University of Utah. Related studies in the lab are funded by National Institutes of Aging of the National Institutes of Health (NIH; 8DP1AG044152), a $2.5 million NIH Director's Pioneer Award that Bass received in 2011. She was one of 13 scientists nationwide to receive the award, one of the most prestigious the NIH gives.
Drosophila Dicer-2 Cleavage Is Mediated by Helicase- and dsRNA Termini-Dependent States that Are Modulated by Loquacious-PD; Niladri K. Sinha, Kyle D. Trettin, P. Joseph Aruscavage, Brenda L. Bass, Molecular Cell (Epub ahead of print); April 16, 2015