Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
What is the Function of Dicer's Helicase Domain?
We are exploring the role of Dicer's helicase domain in C. elegans. We find that the germline defects of C. elegans lacking Dicer (dcr-1(-/-)) are rescued by a transgene encoding wildtype Dicer, as well as by transgenes encoding Dicer with point mutations in the helicase domain. Further, all strains are wildtype in their ability to mount an RNAi response following feeding of exogenous double-stranded RNA (dsRNA).
The finding that the helicase mutants were not deficient for exogenous RNAi led us to assay for defects in endogenous small RNA processing. We assayed piRNAs, 4 miRNAs, and 5 endo-siRNAs by northern blot and saw no noticeable defects in processing either piRNAs or miRNAs. However, helicase mutant lines were completely devoid of endo-siRNAs. We also assayed changes in cognate mRNA levels for four of the missing endo-siRNAs, and observed a corresponding increase in mRNA level. To get a more complete picture of the endogenous small RNA defects in our Dicer helicase mutants, we performed high throughput sequencing of small RNAs from wildtype and helicase mutant rescue strains, using a protocol designed to look specifically at primary endo-siRNAs. Preliminary analyses of these data indicate that the helicase domain is required for the accumulation of many, but not all primary endo-siRNAs. We are currently performing analyses to determine the defining features of endo-siRNA loci that require a functional helicase domain versus those loci that accumulate endo-siRNAs in both wildtype and helicase-defective strains.
Our model is that Dicer's helicase domain allows the enzyme to act processively, binding long dsRNA and cleaving along its length before release. A single double-stranded cleavage is sufficient to generate a miRNA from its short precursor dsRNA, explaining why the helicase domain is not required for miRNA processing. This model is based on in vivo data, and we are now attempting to prove the model with in vitro biochemical studies.
Noah C. Welker
Department of Biochemistry