We identified a background mutation in strain CB3388 ncl-1(e1865) III that causes an RNAi-defective phenotype (Rde) when the strain is tested on lin-29 and nhr-23 feeding RNAi and in unc-54 dsRNA injection assays, but shows normal RNAi response when tested on pos-1 and unc-22 feeding RNAi. We mapped this mutation to LGI ~+4.6. The Rde phenotype was rescued via transgenic expression of B0379, which contains the mut-16 gene (B0379.3) required for RNAi (Vastenhouw et al., 2004). Injection of a PCR product containing mut-16 alone rescued the Rde phenotype. We found no sequence changes in mut-16 coding regions in CB3388, however we found a 451 bp deletion approximately 500 bp upstream of the mut-16 start codon (mg461) (Table 1). mg461 does not confer the germline phenotypes observed in mut-16(pk710) null mutants (e.g. Rde to pos-1 RNAi, High incidence of males (Him), temperature-sensitive (ts) sterile), therefore we hypothesize that mg461 is a hypomorph, affecting only the somatic expression and function of mut-16. We are currently attempting low copy rescue experiments and sequencing all non-coding regions of the mut-16 gene to confirm that no additional lesions exist that may cause the Rde phenotype.

The mg461 deletion is also present in strains XM1011 inx-22(tm1661) I, and XM1012 inx-22(tm1661) I; fog-2(q71) V. Like CB3388, XM1011 is resistant to feeding RNAi targeting somatically-expressed genes including nhr-23, lin-29, bli-1, dpy-7, and unc-45, but sensitive to RNAi targeting the germline-expressed genes pal-1 and pos-1. The Rde mutation in XM1011 failed to complement both mut-16(pk700) and mut-16(pk710), and the mg461 deletion was confirmed by sequencing.

We discovered mut-16(mg461) in the background of two strains from independent C. elegans laboratories. The DNA sequence around the deletion does not contain obvious repeats or transposons that would suggest a high frequency of spontaneous mutation. Furthermore, the lesion is identical in both strains and therefore appears to have come from a common ancestral strain. PCR genotyping suggests that mg461 is prevalent in strains from several labs and may have arisen during the early years of C. elegans research (see Gabel et al., B, this issue of WBG). The presence of mg461 can confound RNAi-based analysis, and poison the discovery of genes that act in small RNA pathways. Because mg461 is not detected by the standard unc-22 and pos-1 Rde assays, and is not strongly Him or ts sterile like many Rde strains, its presence is not obvious to the experimenter. Researchers are encouraged to genotype laboratory “N2” stocks and any strains used in RNAi-based analyses to ensure that the mg461 mutation is not present.