FIGURE 3: Rrp6p and Rrp47p exist as separate complex independent of the core exosome. (A) Workflow depicting the experimental approach involving the reciprocal Co-IP that demonstrates a physical association between Rrp6p and Rrp47p. Cell extracts prepared from the yeast cells (yBD-507) expressing Rrp47-TAP were subjected to immunoprecipitation either with anti-TAP antibody or with anti-Rrp6p antibody followed by detection of the Rrp6p in anti-TAP IP and detection of Rrp47p in anti-Rrp6p-IP. (B) Experimental outline to demonstrate the existence of Rrp6p-Rrp47p complex in the cell extract immune-depleted of Dis3p. Cell extracts prepared from the yeast strain expressing Dis3p-TAP and Rrp47-myc (yBD-540) was subjected to immunoprecipitate with anti-TAP antibody followed by detection of Dis3p in the input, IP, and supernatant fraction using an anti-TAP antibody. After ensuring the absence of Dis3p in the supernatant fraction (right side panel in the top row), it was further subjected to immunoprecipitation with an anti-myc antibody to precipitate Rrp47 followed by the detection for the presence of Rrp6p, Rrp4p (a component of the core exosome) and Mtr4p (a component of the TRAMP complex). The methodology involving cell extract preparation, immunoprecipitation, and western blotting is described in the materials and method section. (C-D) Fractionation/separation profiles of the components of the nuclear exosome, Rrp6/47p and free Rrp6p, Rrp4p, and Rrp47p in Biogel P-200 gel-filtration chromatographic column showing Rrp6p and Rrp47p exists as a separate complex independent of the core nuclear exosome. Cell extracts prepared from the yeast strain expressing Rrp47-TAP (yBD-507) was subjected to fractionation by Biogel P-200 Gel filtration column as described in materials and methods and the eluted fractions were subjected to western blotting analysis using anti-Rrp4p, anti-Rrp6p, and anti-TAP (for detection of Rrp47p). Panel D depicts a qualitative representation of the elution profile of different proteins in the Biogel P-200 Gel filtration column. Relative elution volumes of various MW markers eluted from this column and the fractions in which they appeared are indicated in this profile. (E) Fraction numbers 11 to 18 of the above elution from the Biogel P-200 column were further subjected to separate co-immunoprecipitation by anti-TAP Ab, and further checked with either anti-TAP (for detection of Rrp47p), or anti-Rrp4p and anti-Mtr4p antibody to demonstrate the absence of the core nuclear exosome and the TRAMP complex in those eluates.