Rrp47p is a multimeric complex. (A) Recombinant His(6)-Rrp47p purification. Samples were resolved through a 15% SDS–PAGE gel and visualized with Coomassie blue G250. Lane 1, non-induced cell extract; lane 2, cell extract after 4 h induction; lane 3, Ni-NTA superflow eluate; lane 4, SP-sepharose non-bound fraction, lane 5, SP-sepharose eluate; lane 6, peak fraction from the superdex 200 GF column. The positions of molecular weight markers (in kDa) are indicated on the left. (B) Gel filtration analysis of the SP-sepharose retained fraction. The A₂₈₀ profile is shown, together with the elution volumes of the markers thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.3 kDa). The calibration curve obtained from the molecular weight markers is shown.

Rrp47p binds directly to the N-terminal region of Rrp6p. (A) Rrp47p co-purifies with Rrp6p in pull-down assays. Glutathione-sepharose beads were incubated first with lysate containing recombinant GST–Rrp6p or GST and then with lysate from cells expressing His(6)-Rrp47p or containing the control vector pRSETB. Eluates were resolved through a 10% SDS–PAGE gel and analysed by western blotting using an antibody specific for the His tag. O denotes the top of the resolving gel; X marks the position of the bromophenol blue tracking dye. (B) Schematic of the domain structure of Rrp6p. PMC2NT, exonuclease (EXO) and HRDC domains are indicated as boxes. Deletions within Rrp6p constructs made in this study are indicated by broken lines. (C and D) Western analyses of eluates from pull-down assays performed using different Rrp6p constructs, as in A. Upper panels show western blots of eluates using an anti-GST antibody; lower panels show corresponding results with an anti-His antibody. Multiple bands are visible with the anti-GST antibody due to proteolytic degradation of Rrp6p. The electrophoretic migration of the full-length Rrp6p protein constructs are indicated to the right of the panels.

Rrp47p interacts with nucleic acid when bound to Rrp6p. Glutathione-sepharose beads charged with GST or a GST fusion protein containing the N-terminal region of Rrp6p (GST-Rrp6Δ212-721) were incubated with lysate containing His(6)-Rrp47p or a control extract (pRSETB). After washing the beads, protein complexes were incubated with radiolabelled E. coli tRNA^Phe or a DNA restriction fragment. The non-bound material was removed and bound tRNA or DNA was determined by quantifying the associated Cherenkov radiation. (A) Levels of tRNA retained. (B) Levels of DNA retained. The results shown are the average of three independent assays, the error bars indicating the associated range.

Loss of the N-terminal region of Rrp6p elicits a slow growth phenotype comparable to rrp47-Δ mutants. Growth of isogenic rrp6-Δ, zz-rrp6, zz-rrp6Δ1-213, rrp47-Δ and wild-type strains were compared on selective solid or liquid growth medium. (A) Spot growth assays. Ten-fold serial dilutions of yeast precultures were incubated on solid medium at 26°C (permissive for growth of rrp6-Δ strains) and 34°C (non-permissive for growth of rrp6-Δ strains) and the plates photographed after 4 days. (B) Growth curves. Cultures grown at 30°C were maintained in the exponential growth phase by dilution in pre-warmed medium. The measured increase in OD₆₀₀, expressed as log₁₀ values, is plotted against time.

Rrp47p is a nucleic acid-binding protein. (A) DNA EMSA. Upper panel, 50 ng linearized plasmid was incubated with 2-fold increasing amounts of IMAC-purified, recombinant Rrp47p over the indicated concentration range. Free and bound forms were resolved by agarose gel electrophoresis and detected by Southern blot hybridization (see Materials and Methods section). Lower panel, EMSA in the presence of competitor RNA. Lane 1, free DNA substrate; lane 2, DNA and Rrp47p; lane 3, DNA and mock-purified protein from a pre-induced culture; lane 4, DNA, Rrp47p and 10 μg poly(A); lane 5, DNA, Rrp47p and 10 μg poly(A)–poly(U). (B) Titration of competitor RNA in EMSA. Mixes contained 50 ng DNA, 5 μM Rrp47p and 2-fold increasing amounts of RNA over the indicated concentration range. Upper panel, poly(A); Lower panel, poly(A)–poly(U). (C) RNA EMSA with poly(A) (left) or poly(A)–poly(U) (right). One hundred nanogram of RNA was incubated 2-fold increasing amounts of Rrp47p over the indicated range. The mixtures were resolved by agarose gel electrophoresis and the poly(A) RNA was detected by northern blot hybridization.

Residues C-terminal to the PMC2NT domain contribute to Rrp47p interaction. (A) Schematic of the N-terminal region of Rrp6p. The location of the PMC2NT domain and the four short α-helices α1–α4 are shown. Residues P176 and L197 define the C-termini of truncated Rrp6p constructs. These residues are mutated to stop codons and therefore not the C-terminal residues. (B) Pull-down assay of His(6)-Rrp47p with GST-tagged N-terminal Rrp6p fragments. Eluates from glutathione-sepharose beads were resolved by SDS–PAGE and assayed by western blot analyses with primary antisera against GST (upper panel) or the His tag (lower panel). Electrophoretic mobilites of protein molecular weight markers (in kDa) are given on the left.

Rrp47p interacts with the N-terminal region of Rrp6p in yeast. (A–D) Western analyses of cell extracts from strains expressing epitope-tagged forms of Rrp47p and/or Rrp6p. Extracts were resolved through 10% SDS–PAGE gels, protein transferred to western blots and the fusion proteins detected using PAP or GST-specific antisera. Relevant strain genotypes are indicated above each lane. The rrp47-zz, rrp6-TAP and zz-rrp6 alleles encode full-length fusion proteins, whereas the zz-Rrp6Δ1-213 and the GST-Rrp6Δ212-721 proteins are truncated Rrp6p variants. The GST fusion proteins were expressed under the control of the GAL promoter; panel D shows analyses of extracts from strains grown in glucose-based media (glc) and in galactose-based media (gal). The upper panel in D shows a western analysis using the GST-specific antibody. The centre panel in D shows a western analysis of the same samples using the PAP antibody. Bands corresponding to the detected fusion proteins are indicated on the right. SDS–PAGE analyses (lower panels) indicate the relative loading of each extract. (E) Western analysis of lysates from isogenic wild-type and rrp47-Δ strains, using a His(6)-Rrp47p antiserum. Proteins were resolved through a 15% SDS–PAGE gel. The migration of size markers (in kDa) is indicated on the left. A strong band of the expected size is detected in the wild-type lysate (lane 1) and absent in the rrp47-Δ extract (lane 2). (F) Western analyses of the bound fractions of extracts from strains expressing GST (lane 1) or GST-Rrp6Δ212-721 (lane 2) after incubation with glutathione-sepharose beads. The upper panel shows a blot probed with the GST-specific antibody, the lower panel shows a blot probed with the His(6)-Rrp47p antiserum.

The N-terminal region of Rrp6p is required for 5.8S rRNA and snoRNA processing. Northern blot analyses of RNA in the zz-rrp6Δ1-213 mutant. Total cellular RNA from yeast cultures was resolved through 8% denaturing acrylamide gels and analysed by northern blot hybridization using probes specific to 5.8S rRNA and snR38 RNA species. Relevant strain genotypes are indicated above each lane. Detected RNA species are indicated to the right of each panel. (A) Hybridization with probes to 5.8S rRNA species. Upper panel, hybridization with a probe (237) specific for 3′ extended 5.8S rRNA species. Centre panel hybridization with a probe (236) specific for the mature 5.8S rRNA. 5.8S rRNA is expressed as a predominant short form and a less abundant long form. Lower panel hybridization with a probe (242) complementary to the RNA pol III transcript SCR1, which serves as a loading control. (B) Hybridization with a probe (272) specific for snR38. Lower panel, exposure showing the mature snR38 and a species with a short 3′ extension (snR38 + 3). Upper panel, longer exposure to reveal 3′ extended precursors. The snR38 species extended at the 3′ end to the 3′ splice site is indicated as snR38-3′.