Figure 4. Mutator Complex Containing Catalytically Dead MUT-14 IsCompetent for siRNA Formation In Vitro(A) Expression and localization of GFP derived from a
mut-16 transcriptionalfusion (
mut-16prom::GFP) and a
mut-16 translational fusion (
mut-16::GFP)in adult animals. Mutator foci and muscle and vulval cells are indicated.(B) Western blot analysis of MUT-16::GFP, FLAG::RRF-1, and HA::EGO-1from cell lysates (in) and anti-GFP coimmunoprecipitates (IP).(C) Top: in vitro assay for siRNA synthesis. Western blot analysis of free GFP(
mut-16prom::GFP) and GFP and mCherry fusion proteins (
mut-14::GFP; mCherry::
rrf-1;
mut-14 smut-1 and mut-14DKAD::GFP; mCherry::
rrf-1;
mut-14) from cell lysates (in) and anti-GFP coimmunoprecipitates (IP). Afaint background band is observed in each of the input samples after probingwith mCherry antibody. Bottom: In vitro synthesized siRNAs followingcoimmunoprecipitation of mutator complexes containing wild-type MUT14(
mut-14::GFP; mCherry::
rrf-1;
mut-14 smut-1) or catalytically deadMUT-14 (mut-14DKAD::GFP; mCherry::
rrf-1;
mut-14). Immunoprecipitatedprotein complexes were incubated with a mixture of radiolabeled and nonlabelednucleotides and in the presence or absence of an in vitro-transcribedmRNA (B0250.8).(D) Schematic depicting siRNA formation by the mutator complex in thegermline.