Structure of RNAPI. Schematic representation illustrating the presumed positions of five unshared RNAPI subunits and their spatial relationship to each other. The model is based on electron microscopy and immunolocalization data published by Schultz and coworkers (4, 11).

Rnt1p interacts with RNAPI-specific subunits. (A) Schematic representation of the Rnt1p truncations and point mutations used in this study. The hatched boxes represent the Rnt1p N-terminal (N-term) domain. The Rnt1p nuclease domain (NUCD) is light gray, and its dsRBD is dark gray. The portion of the dsRBD in black represents the Rnt1p protein-binding site. The white box indicates the Rnt1p nuclear localization signal (NLS). The positions of the different point mutations are indicated by asterisks. The sequence of the C-terminal 51 aa of Rnt1p and the positions of the different mutations in this region are shown at the top, as well as the extents of α-helices 2 and 3 of the dsRBD. (B) The interaction between RNAPI subunits Rpa12p and Rpa34p with the different domains of Rnt1p was assessed by analyzing the abilities of plasmid pairs to support the growth of yeast strain PJ69-4A on medium lacking histidine and supplemented with 10 mM 3-aminotriazole (YC −His + 10 mM 3AT) compared to growth on nonselective medium (YC −3). (C) The two-hybrid assay was carried out as described for panel B, with Gar1p as bait, in order to evaluate the specificity of Rnt1p interaction with the RNAPI subunits. (D) RNAPI subunits Rpa34p and Rpa49p coimmunoprecipitate with TAP-tagged Rnt1p (Rnt1p-TAP). Immunoprecipitations with cell extracts expressing either tagged or untagged Rnt1p were performed, and the Rnt1p-bound proteins (IP) were released by enzymatic cleavage of the Rnt1p tag (Rnt1p-C). The released proteins were visualized by Western blot analysis with protein-specific antibodies. Antibodies against phosphoglycerate kinase (PGK) were used as a control. The Rnt1p complex was treated with different concentrations (units) of a mixture of RNases V1, T1, and T2 in order to evaluate the contribution of RNA to the formation of the protein complex.

Deletion of RNT1 inhibits rRNA synthesis. (A) The rate of rRNA synthesis was determined by following the incorporation of [³²P]PO₄ in both pre-rRNA and mature rRNA species. Labeled RNAs separated on denaturing agarose gels were quantified and plotted using the amount of total stained RNA as a reference point. The data presented are an average of three experiments. (B) Pulse-labeling of rRNA upon the deletion of RNT1. Wild-type and Δrnt1 cells were incubated for either 15 or 90 min with [³²P]PO₄ at 26°C. The RNA was extracted, and equal counts were loaded on an agarose gel and visualized by autoradiography. To evaluate the RNA quality, equal amounts of total RNA were loaded onto agarose gel and visualized by direct staining. The positions of the different RNA species, as well as of the degradation products (DP), are indicated on the left. (C) Chart illustrating the ratio of rRNAs quantified after 90 min of labeling with a PhosphorImager. The expression level of 35S rRNA relative to that of mature rRNA was calculated and normalized to that of wild-type RNA. (D) Pulse-labeling of rRNA upon the inactivation of Rnt1p. Cells expressing a temperature-sensitive allele of Rnt1p (rnt1-ts) were grown for 0, 1, or 240 min at 37°C and then incubated with [³²P]PO₄ for 5, 15, or 90 min. The RNA was extracted and visualized as described for panel B. DP indicates a degradation product observed upon the inactivation of Rnt1p. (E) Chart illustrating the relative expression levels of the rRNA species detected after 90 min of labeling. The RNA values were normalized with the RNA extracted from rnt1-ts cells growing at the permissive temperature as a reference.

Deletion of RNT1 delays rRNA processing. Wild-type (H1227) and Δrnt1::HIS3 cells were pulse-labeled for 2 min with [³H]uracil, followed by a chase with excess unlabeled uracil. Aliquots of the cultures were collected at the indicated time points starting from the beginning of the chase. RNA was extracted and separated on denaturing agarose gels and transferred to nylon membranes. The observed rRNA species are indicated on the left.

Loss of Rnt1p activity increases the number of rRNA genes with an open chromatin conformation. (A) Cells carrying a wild-type copy of RNT1 or a temperature-sensitive allele (rnt1-ts) were shifted to 37°C, and aliquots were collected at different time points for RNA extraction or nucleus preparation and psoralen photo-cross-linking. Southern blot analysis of the cross-linked DNA (top) was performed with a probe corresponding to the 25S rRNA. The open and closed rRNA gene forms are indicated by open and black circles, respectively. The arrowhead on the right indicates the open form of the rRNA genes. The bottom part shows an RNase protection assay performed to assess the impact of Rnt1p inactivation on the processing of the 25S rRNA 3′ end (B0) as a control. The values on the right are molecular sizes in nucleotides. (B) The psoralen-cross-linked rRNA gene shown in panel A was quantified with a PhosphorImager, and the results are presented as migration profiles. The profiles of RNT1 DNA are in black, while those of rnt1-ts DNA are in gray. (C) Ribosomal protein Rpl15Ap, 5′-end processing factor Utp7p, and Utp5p, a member of the t-Utp complex, were expressed from tetracycline (TetO₇)-controllable promoters, and the rRNA genes forms were examined after inhibition of the transcription of each gene. Psoralen cross-linking and Southern blot assays (top) were prepared as described for panel A, and the migration profiles (middle) of the cross-linked DNA were prepared as described for panel B. The migration profiles of DNA extracted after 0 and 24 h of transcription inhibition are shown as black and gray lines, respectively. The amounts of 27S pre-rRNA and 18S rRNA were examined by Northern blot analysis after 24 h of transcription inhibition, and the RNA amount was quantified and plotted relative to the amount of 5.8S rRNA (bottom). WT, wild type. (D) Psoralen cross-linking was performed with nuclei extracted from wild-type cells (RPA12) or cells lacking RPA12 (rpa12Δ). The Southern blot assay (left side) was prepared as described for panel A, and the migration profile (right side) was prepared as described for panel B. The profile of RPA12 DNA is in black, and that of rpa12Δ is in gray.

Mutations that disrupt the Rnt1p/RNAPI complex increase the number of open rRNA genes and reduce the efficiency of pre-rRNA processing. (A) The ratios of open to closed rRNA genes were examined in cells expressing different mutations that impair Rnt1p catalytic activity (GFP-D247/R), block the localization of Rnt1p in the nucleolus (GFP-463st), or disrupt the interaction with RNAPI (AD-463st and BD-K421/A). The open and closed rRNA genes are indicated by open and black circles, respectively, on the left. The profiles corresponding to the samples shown in panel A are presented in panel B. Wild-type profiles are in black, while those of the different mutants are in gray. (C) Northern blot analyses of rRNAs extracted from cells expressing the different Rnt1p mutants examined in panel A. The pre-rRNA processing intermediates were visualized with probes against sequences in the 5′ ETS, ITS1, 5.8S rRNA, or 3′ ETS (I to IV). Schematic representations of the primary rRNA transcript and probe positions are shown at the top. The positions of the different processing intermediates are indicated at the sides, with asterisks denoting 3′-extended species detected with probe IV.