Harvard Team Applies RNAi to RNAi To Uncover RNAi-Related Genes

About two years ago, John Kim, a postdoctoral fellow in the lab of Gary Ruvkun at Harvard Medical School, was looking for a better way to study RNAi on a genome-wide level. What he came up with was a novel method of applying RNAi to RNAi.

Together with colleagues including Harrison Gabel and Ravi Kamath, he used this approach to screen the C. elegans genome to find genes required for RNAi. The results of their work were published last week in Science.

"The idea was to have a very sensitive assay to indicate when RNAi stops working in an organism, in particular C. elegans," Kim told RNAi News this week. "I thought, 'Wouldn't it be cool to use RNAi to inactivate RNAi genes?'."

To do so, Kim developed an "RNAi sensor" strain of C. elegans that expressed a green fluorescent protein dsRNA hairpin and a GFP reporter gene in C. elegans epithelial seam cells. The researchers found that GFP expression was robustly restored in worms fed RNAi sensor strain bacteria that express dsRNA corresponding to genes. In comparison, GFP expression remained inactive in worms fed control dsRNA, according to the Science paper.

"I'm sure other people have thought of [this] before, but it [involves] a sort of circular logic because you are using RNAi to kill RNAi," Kim said. "I suspect people were discouraged just from a logical standpoint."

Kim did note that Bob Goldstein and colleagues at the University of North Carolina published in 2002 a paper in the Proceedings of the National Academy of Sciences describing how they co-injected dsRNAs into C. elegans to identify RNAi-related genes. However, Kim stressed that his work began before the PNAS paper was published, and noted that Goldstein's approach "is simply not a high-throughput method of studying RNAi" because of the vast number of injections that would have to be required for a genome-wide project.

After getting encouraging results when they applied the RNAi sensor to a select group of C. elegans genes, Kim and his colleagues decided to expand their experiments to the entire genome. This broad screen identified 90 genes that impact RNAi. Eleven of the genes correspond to loci known to be required for RNAi, including the core RNAi machinery such as dcr-1, rde-1, and rde-4, the researchers wrote in the Science paper.

Meanwhile, "54 of the genes are essential for viability and one-third of the viable 25 new genes exhibit reduced brood sizes," the paper's authors stated. Additionally, 85 percent of the new genes have human homologs, suggesting conserved functions.

"It is possible that some of the identified factors could be non-specific; for example, inactivation of a factor … could inhibit the expression from one epidermal promoter of the RNAi sensor strain but not the other," the authors said in the paper. "However, since a large majority of the RNAi clones tested also affect transgene silencing in a variety of other tissues, most are likely to act in the RNAi pathway."

The results of the screen were for Kim and his colleagues a validation of sorts of their long-held belief that RNAi was more important to life than many give it credit for. "We have always held the view that RNAi would be essential for life in an organism," he said. "Our belief is that these have eluded detection because of the genetic schemes in which RNAi has been studied. So when we found that 54 of our genes are essential for life, that supported our view.

"Of the other ones that allow for viable progeny, many of them give smaller brood sizes," he added. "So the questions are now: 'What do the genes do? What sort of pathways does RNAi function in? Those are just wide open at this point."

Among some of the other findings from the C. elegans screen as detailed in the Science paper:

• RNA binding and processing factors comprised the largest class of new RNAi factors the researchers identified, and these suggest "new steps in the RNAi pathway as well as overlap with other RNA-mediated gene regulatory pathways."
• The researchers identified the nonsense-mediated decay gene smg-2 — as well as three genes predicted to function in NMD, T25G3.3, paa-1, and F26A3.2 — as modulators of RNAi, consistent with previous observations implicating smg-2, and, to a lesser degree, smg-5 and smg-6, in RNAi.
• Among the new RNAi candidates identified were six proteins with domains found in known RNAi factors: two proteins contain either a PIWI or both PIWI and PAZ domains found in Argonaute and the RNAi factor RDE-1; F22D6.6 possesses a Tudor RNA-binding domain identified in the TSN micrococcal nuclease of RISC; and Y38A10A.6, F56D2.6, and C06E1.10 have DEAD/DEAH-box motifs found in Dicer, MUT-14, DRH-1, and DRH-2.
• "Other classes of factors that regulate RNAi include DNA repair factors RuvB and Brca2, and the MAP kinase pathway factors ZC449.3 and MTK-1. The DNA repair and recombination factors suggest that the RNA replication or TGS steps in RNAi may include checkpoints and control mechanisms related to those used in DNA replication and recombination."
• "Transgene silencing in C. elegans is mechanistically related to RNAi. A subset of genes required for RNAi are essential for transgene silencing in the germline, including dcr-1, mut-7, and mut-16. However, other genes including rde-1 and rde-4 are essential for RNAi but dispensable for germline silencing demonstrating that germline gene silencing and RNAi may require distinct, possibly paralogous, sets of genes."
• RNAi-inactivation of each of the 90 RNAi candidates did not affect precursor microRNA processing, which underscores the lack of overlap between RNAi and the microRNA pathways at this step. The researchers looked at phenotypes associated with defects in the heterochronic pathway controlled by the let-7 microRNA, mutations in which cause supernumerary seam cells. "Of the 90 RNAi factors, inactivation of six genes, including dcr-1, caused an increased number of seam cells and, of those, only three (dcr-1, pop-1, and kin-10) also significantly enhanced the weak let-7 protruding vulva phenotype. Furthermore, pop-1 RNAi may cause cell fate transformations rather than microRNA defects. These findings indicate little molecular overlap between the new RNAi factors and factors required for the microRNA pathway."

  Kim stressed, however, that "we might not have sensitive enough or specific enough assays to see if [RNAi factors] are involved in the microRNA pathway. We do believe a small number of the genes may play a role in microRNA pathways," he said.

— DM