Figure 3.

SART3 and USIP1/ZK863.4 are in a U6 snRNA-containing complex. (A) Western blot of anti-FLAG co-IP of C-terminally GFP/3xFLAG-tagged USIP-1 (lanes 1 and 3–5) and a C-terminally His/FLAG-tagged GFP control construct (lanes 2 and 6–8). USIP-1/GFP/3xFLAG and GFP/His/FLAG constructs were detected with anti-GFP. Endogenous SART3 was detected with a polyclonal antibody against SART3. Two percent of input and 70% of IPs were loaded on a gel. (B) Proteins identified by mass spectrometry following IP of FLAG-tagged SART3, USIP-1 or GFP. The latter serves as a negative control (see additional proteins in Supplementary Table S2). Numbers of spectra mapping uniquely to a given protein are indicated. (C) Northern blot of RNA extracted from eluates obtained by an anti-FLAG co-IP on lysates from worms expressing the indicated transgene. Two milligram total protein was used as input for the co-IP and 100% of the co-immunoprecipitated RNA was loaded on the gel. 2.5 μg of total RNA were loaded as a reference. Subsets of probes were applied to the membrane to detect different RNAs simultaneously; the asterisk indicates an unspecific band detected with the probe against U6. (D) Fluorescence and DIC microscopy of L4 stage worms expressing C-terminally GFP/3xFLAG-tagged USIP-1 from a fosmid. Arrows point to nucleoplasm, arrow heads point to nucleolus. Scale bar, 20 μm.