The nuclear poly(A) polymerase and Exosome cofactor Trf5 is recruited cotranscriptionally to nucleolar surveillance

Localization of SSU-proccesome components at the 5′ end of the 25S gene is dependent upon ongoing transcription and the physical presence of nascent transcripts. (A) Depletion of Rpa43 severely impacts the distribution of Rpa190 along the rDNA. (Upper panel) Growth curves. tet::rpa43 cells were grown in complete medium to mid-log phase (OD₆₀₀ ∼ 0.3), doxycycline (Dox) was added at a final concentration of 10 μg/mL, and growth followed by OD₆₀₀. (X axis) time in Dox (hours); (Y axis) OD₆₀₀/OD₆₀₀ at 0 min expressed in a log scale. (Lower panel) Distribution of Rpa190 along the rDNA in the presence and in the absence (+Dox) of Rpa43. Depletion of Rpa43 was achieved following 12 h of transfer to doxycycline-containing medium (see arrow, panel A). Levels of Rpa190 interaction with the rDNA at each position were tested as described in Figure 1. Amplicons color-coded and numbered as in Figure 1. (B) In the presence of reduced recruitment of Pol I, SSU-processome components do not interact at the 5′-end of the 25S gene (amplicon 11). (Upper panel) The presence of Utp4, Utp7, and Utp9 at the 5′-end of the 25S gene was tested by Ch-IP in the presence of either physiological (no Rpa43 depletion) or strongly reduced Pol I recruitment (+Dox, corresponding to 12 h depletion of Rpa43, see panel A). (Lower panel) Effect of Rpa43 depletion on the steady-state level of Rpa190, Utp4, Utp7, and Utp9. Equivalent amounts of chromatin, according to cell OD₆₀₀, extracted in the presence and absence (12 h depletion time point) of Rpa43, were analyzed by SDS-PAGE and processed for ECLplex. As a control for loading, membranes were probed for G-6-PDH. The amount of target protein was normalized to G-6-PDH and set to 1 in the presence of Rpa43. (C) In the absence of RNA, SSU-processome components do not interact at the 5′-end of the 25S gene. Distribution of Utp1 (UTP-B) and Utp9 (UTP-A) along the rDNA locus in the presence and in the absence of RNA. Chromatin extracts were treated (+RNase) or not with RNase prior to coprecipitation and qPCR analysis (see Materials and Methods).