Pre-rRNA and rRNA are stabilized upon TRAMP and Exosome inactivation in strains depleted for SSU-processome components. Total RNA was extracted from cells grown to mid-log phase in complete medium, separated in denaturing agarose gels, and transferred to nylon membranes for Northern blot hybridizations. RNA was extracted from an otherwise isogenic wild-type strain, from strains deleted either for the TRAMP component Trf5, the Exosome cofactor Rrp6, or for both (lanes 1–4,45–48), as well as from cells depleted for Utp5 (lanes 5–24) or Utp11 (lanes 25–44), either in the presence or in the absence of functional TRAMP and/or Exosome complexes. UTP depletion was achieved in tet:: regulated strains following transfer to doxycycline-containing medium for the time points indicated (6–24 h). A description of the pre-rRNA processing pathway in yeast, as well as the oligonucleotide probes used in the hybridizations, is provided in Supplemental Fig. S1. Panel I was hybridized with probe b; panel II with c, panel III with a and f, and panel IV with e. As a loading control, membranes were hybridized for SCR1, the RNA component of SRP (panel V).
