Several components of the nucleolar surveillance co-localize with nascent pre-ribosomes. (A) rDNA Ch-IP analysis of components of the TRAMP (Air1, Air2, Trf4,Trf5, and Mtr4) and Exosome (Rrp44 and Rrp6) complexes. (Upper panel) Yeast strains expressing a functional carboxyl-terminal TAP-tagged fusion of Air1, Air2, Mtr4, Rrp6, Rrp44, Trf4, and Trf5 and wild-type isogenic control (TAP) were analyzed by Ch-IP and qPCR as described in Figure 1. (Lower panel) Western blot analysis of protein coprecipitation efficiency (see legend to Figure 1). (B, upper panel) Trf5 is recruited cotranscriptionnally to the rDNA. Ch-IP analysis was performed in the presence and in the absence of Rpa43 (+Dox, 12 h depletion) (see Fig. 2B). (Lower panel) Effect of Rpa43 depletion on the steady-state level of Trf5 (see Fig. 2B). The depletion of Rpa43 resulted in a mild reduction in the steady-state level of Trf5 of about 15%, the concurrent decrease in the level of interaction with the rDNA was, however, twofold. (C) In the absence of RNA, Trf5 does not interact at the 5′-end of the 25S gene. Chromatin extracts were treated (+RNase) or not with RNase prior to coprecipitation and qPCR analysis (see Fig. 2C).
