Mark Blaxter's teaching pages

Functional Genomics in C. elegans 3:

RNAi    RNA mediated interference

(1: Microarray analysis 2: Expression analysis using reporters 3: RNAi)

Ketting RF, Haverkamp TH, van Luenen HG, Plasterk RH Cell 1999 99:133-41 mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.

C. elegans can be made transgenic by injection of DNA into the syncytial gonad.

Early attempts at knockdown of gene fuction by

antisense RNA injection

transgenesis with antisense-producing coonstructs

met with limited and/or variable success.

In some labs, antisense RNA injection gave reliable and significant impairment of gene fuction: in cases where null mutations were known, the phenocopy was equivalent to null.

In other labs either no phenocopy or partial effects were noted.

The Fire and Mello labs noted that in some labs both "sense" and "antisense" RNA injection gave phenocopies.

It turns out that the labs injecting "pure" antisense RNA were getting poorer results than those injecting mixtures of sense + antisense (due to lack of prevention of antisense/sense readthrough in their constructs). Careful preparation of sense and antisense strands revealed that both were needed: the phenomenon is thus dsRNA dependent.

Other Features of RNAi

Fire, Mello and coworkers also showed:

that it was not necessary to inject the gonad: injection of the body cavity or the gut lumen resulted in significant RNAi

that feeding the nematodes on E. coli expressing dsRNA was sufficient to get phenocopies

that soaking the nematodes in dsRNA would also give phenocopies

that the effect was achieved only with transcribed gene segments, and that exons were much more effective than introns

that the effect appeared to be on mRNA levels: RNAi reduces or ablates steady state RNA levels for cytoplasmic and nuclear transcripts

that many different genes could be affected: early development genes, maternally or zygotically transcribed genes, or late, "differentiation product" genes (such as muscle proteins)

that endogenous, chromosomal genes and genes in extrasomal transgenic arrays could be affected

that the phenocopy was "transmissable" in that it was seen in the F2 and F3 progeny of injected mothers. The eff3ects did lessen with generational passage, in both individual expression and in the proportion of animals expressing the phenotype

that dsRNAi also works in other species, from protozoa to flies.

Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC

Cell 1999 99:123-32

These authors sought to identify the nematode genetic machinery behind RNAi by identifying mutants resistant to a lethal RNAi: the knockout of function of a gene called pos-1. Four rde loci were identified. The mutations can be split into 2 kinds:

those with "mutator" (elevated levels of transposon mobilisation) phenotype

those with wild type levels of transposon mobilisation

Many transgenes are silenced in the germline, a phenomenon that appears to be associated with the structure of the transgenic DNA (number of copies and arrangement of the transgene). mut-7 is effective at reversing germline silencing. rde-1 is not.

The effects of the rde genes is required in the target tissue, as the mutant animals appear to be wild type for the transport of dsRNA and its localisation in the germ line.

rde-1 was cloned. The rde-1 gene appears to be inessential for growth in the lab, as one of the mutations is a deletion of the coding region.

rde-1 defines a family of 23 C. elegans genes, and a number of homologues from other species implicated in germline maintenance and developmental regulation. Mutation of a Drosophila homologue, zwille, causes derepression of a silent multicopy gene Stellate.