Figure 1.
nrde-1 encodes a nuclear-localized protein that is required for nuclear RNAi. (A) Genetic map positions of genes identified in genetic screen. Number of alleles identified in screen are indicated. (B) RNAi-driven transcriptional inhibition requires
nrde-1 and
nrde-4. Nuclear Run On (NRO) analysis of transcription from the
lin-15b/a gene from nuclei of animals exposed to +/2
lin-15b dsRNA or +/2 a-amanitin. Data are expressed as a ratio +/2
lin-15b dsRNA (normalized to transcription detected from
eft-3 gene) or +/2 a-amanitin (5 mg/ml). Dotted line indicates a ratio of 1; i.e. no change. The genetic background for this experiment was
eri-1(
mg366). Control +/2
lin-15b dsRNA (n = 3-8, +/2 s.e.m.),
nrde-1(
gg088) (n = 4-6, +/2 s.e.m., data point represented by triangle n = 1),
nrde-4(
gg129)(n = 5, +/2 s.e.m.), control +/2 a-amanitin (n = 3 +/2 s.e.m., data point represented by triangle n = 1). D= fold change. Below: diagram of
lin-15b/lin-15a gene structure indicating location of primers and trigger dsRNA (magenta). (C) Predicted
nrde-1 gene structure. Arrows indicate mutant alleles. (D) NRDE-1 localizes to the nucleus. Fluorescence microscopy of two seam cells in a L4 larval animal expressing gfp::
nrde-1. Arrows indicate nuclei. (E) gfp::
nrde-1 fusion gene rescues
nrde-1 mutant phenotype. Animals of the indicated genotypes were exposed to
lir-1 dsRNA. A score of 5 indicates all animals died during larval development and a score of 0 indicates animals did not exhibit any developmental defects. Plates were scored blind and in triplicate for
lir-1 RNAi-mediated lethality (a.u. arbitrary units).