Elevate me from my ignorance: what is known about RNAi of off-target genes that share an operon? Or to be more practical, If I am targeting a gene in an operon with an RNAi feeder clone, might I also knock down to some extent the other transcripts on that operon?
Any info would be appreciated, especially directing me to references…
Different results have been reported for different operons; RNAi of one gene can target other genes in the same operon, but does not necessarily do so.
I’m not knowledgeable about the more recent literature, but I’d point you to the first such report that I know of, a paper from Michel Labouesse’s group reporting that RNAi targeting lir-1, or even lir-1 introns, can apparently affect the function of lin-26, in the same operon, although loss of lir-1 function or RNAi targeting the lir-1-related gene lir-2 has no similar effect. You could do worse than to start with that paper and the papers that cite it.
Scott Kennedy’s lab presented on this topic at the international worm meeting. From what I remember he was working on the genes involved in nuclear RNAi using operons. You should contact him.
RNAi one gene in an operon can knock down the expression of another gene of the same operon at the pre-mRNA level. From Andy Fire’s 1998 paper, they targeted lin-15b exon 7 region and got 2% Muv. Michel Labouesse’s group reported similar thing in the lir-1/lin-26 operon.
We test this by feeding dsRNA to eri-1 animal (enhanced RNAi). We got >90% Muv by targeting exon V of lin-15b, and 100% lethal of lir-1 dsRNA.
We have mutants specifically block this process, but not canonic RNAi processes.
However, we did not see this operon effect on every operon we tested. We picked up RNAi bugs targeting operons from the Ahringer library, and tested whether they gave to different phenotypes by comparing N2 and NRDE mutants. Only several RNAi show desired differences. In that case, we do not think it is a big problem for normal RNAi experiments. However, if you worry about this, we can send you the NRDE-3 mutant.