Figure 2 OMA-1 is a component of riboncleoprotein particles (RNPs). (A) OMA-1::S::TEV::GFP (asterisk) is depleted after incubation with matrix-coupled anti-GFP antibodies (compare lanes 1 and 3). OMA1::S (double asterisk in A-C) is subsequently eluted from the affinity matrix by digestion with TEV protease. A total of 0.25% of each lysate and 1% of each TEV-eluted sample were loaded. Purifications and protease cleavage were monitored by western blotting using either anti-OMA-1 (shown), antiS-tag, or anti-GFP antibodies. Here, and in subsequent panels, samples marked with a plus (+) sign were prepared from lysates containing OMA-1::S::TEV::GFP. Samples marked with a minus (2) sign are negative controls prepared from lysates lacking OMA-1::S::TEV::GFP. All purifications were performed in an
oma-1(
zu405te33) genetic background and were from
fog-1(ts) females, unless otherwise specified. (B) Abundant proteins that copurify with OMA-1::S from
fog-1 and
spe-9 extracts were visualized by staining a polyacrylamide gel with SYPRO-Ruby (red boxes). Proteins in negative controls [minus (2) sign] are similar in size to human keratins (Moll et al. 2008), common contaminants of protein purifications. (C and D) Many proteins require RNA for their association with OMA-1. (C) Treatment with RNase A, prior to incubation with TEV protease (RNase elution, r+), causes proteins to elute from the immunoaffinity matrix. Proteins are not eluted by a mock RNase treatment (RNase elution, nr). Comparatively few proteins copurify with OMA-1::S after RNase A treatment (TEV elution, r+). Proteins were visualized using SYPRO-Ruby. (D) Western blots show that CGH-1 and CAR-1 copurify with OMA-1::S in an RNA-dependent manner. (E-J) OMA-1 reorganizes into large RNPs (arrowheads) when the sperm-dependent signal promoting meiotic maturation is absent or not transmitted to oocytes. Oocytes expressing the rescuing OMA-1::S::TEV::GFP fusion protein show a diffuse pattern of GFP localization (E and F), similar to
spe-9(ts) animals raised at 25 (Figure S1). If sperm are absent, as in
fog-1(ts) animals raised at 25, OMA-1::S::TEV::GFP reorganizes into large foci (G and H). Similar foci form in the presence of sperm when the MSP-dependent signaling pathway active in sheath cells is disrupted, as in
acy-4(
ok1806) mutants (I and J). Explicit genotypes are specified in the legend to Figure S1. Bar, 20 um.