Cofactors of the exosome and TRAMP complexes play different roles in 5′ETS fragment and aberrant pre-rRNA degradation. (A) HeLa cells were depleted of various subunits and cofactors of the exosome and TRAMP complexes. For PAPD5, mRNA levels (normalized to GAPDH) were measured using qPCR. Proteins extracted from control cells and those depleted of POLS were separated by SDS-PAGE and analyzed by Western blotting. (B) RNAs from HeLa cells described in A were analyzed by agarose-glyoxal gel electrophoresis, followed by Northern blotting using a probe hybridizing between the A′ and A0 cleavage sites (ETS2). Note that the increased signal seen for ETS2 in the DIS3 lane is due to the fact that this lane is slightly overloaded relative to the control. (C) RNA from control cells or those depleted of RRP6 by RNAi was analyzed by Northern blotting using probes hybridizing in the 5′ETS (ETS1, ETS2) and at the 5′ end of 28S (28S1302, 28S1727). (D) Schematic representations of the full-length pre-rRNA transcript and the aberrant 37S* pre-rRNA. The positions of additional probes used in Northern blotting are indicated above the 47S pre-rRNA.
