Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs

Abstract

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

FIG. 1.

Rrp47p is a novel exosome-associated factor. (A) Identification of Rrp44p-associated proteins. SDS-PAGE analysis of protein eluted from immunoprecipitated zz-Rrp44p was carried out. Lanes 1 to 10, linear 0 to 2 M MgCl₂ gradient in TMN-150 buffer; HAc lane, postgradient eluate in 0.5 M acetic acid. Proteins were visualized by staining with Coomassie G-250 and identified by mass spectrometry. Rrp47p was eluted in lanes 1 to 2 (∼0.2 to 0.4 M MgCl₂). (B and C) Rrp47p is associated with Rrp4p and Rrp6p. Western blot analyses of immunoprecipitates from wild-type (lane 1), Rrp44p-TAP (lane 2), and Rrp47p-zz (lanes 3 to 4) strains, with antisera specific to Rrp4p (B) and Rrp6p (C) were done. Rrp47p-zz immunoprecipitates were eluted with or without prior digestion with RNase A. As controls for the specificity of the Rrp4p antiserum, cell extracts were loaded from strains expressing wild-type Rrp4p (B, lane 5) or epitope-tagged His (6)-Rrp4p (B, lane 6). Cleaved Rrp6p-TAP was included as a positive control for the Rrp6p Western analysis (C, lane 5). This protein is predicted to migrate slower than enodogenous Rrp6p in SDS-PAGE gels due to the presence of the calmodulin-binding domain.

FIG. 2.

Rrp47p is required for early pre-rRNA processing events. (A) Organization of the yeast pre-rRNA and processing pathway. The 18S, 5.8S, and 25S rRNAs are separated by internal transcribed spacers ITS1 and ITS2 and flanked by the external transcribed spacers 5′ETS and 3′ETS. Coding regions are indicated by thick bars; spacer regions are indicated by thin lines. Sites within the pre-rRNA complementary to probes used in the present study are indicated. Early cleavages at sites A₀, A₁, and A₂ generate the 20S and 27SA₂ pre-rRNAs. The 20S pre-rRNA is processed to 18S rRNA by cleavage at site D. 27SA₂ is processed in ITS1 and ITS2 to generate the 5.8S and 25S rRNAs. Two forms of 27SB, 7S, and 5.8S that differ by 7 nt at their 5′ ends (boxed) are generated by alternative processing pathways. The 23S and 21S pre-rRNAs arise through premature cleavage at site A₃. The 17S′ species results from 5′ degradation of pre-rRNA blocked for processing in ITS1. (B) Northern blot analyses of pre-rRNAs from wild-type, rrp47-Δ, rrp6-Δ, and rrp4-1 strains during growth at 30°C and after transfer to 37°C for 3 h. The probes used are given in brackets to the left of each panel; the species detected are indicated to the right (see panel A). The 23S* species detected with probe 002 is more abundant than 23S detected with probe 003 due to the limited 3′ degradation of 23S pre-rRNA in the rrp4-1 mutant grown at 37°C. (C) Primer extension analyses of 27S pre-rRNAs with probe 006. Extension products corresponding to 27S species processed to sites A₂, A₃, B_1L, and B_1S are indicated. RNA from a GAL::nop7 mutant grown in YPD medium for 24 h is included to provide a marker for the 5′ end of 27SA₃ (29).

FIG. 3.

Rrp47p is required for 7S pre-rRNA processing and 5′ETS degradation. (A) Northern blot analyses of 5.8S rRNA species in a wild-type strain and in exosome mutants. RNA was recovered from wild-type and mutant strains, as in Fig. 2. (Upper panel) Hybridization with a probe specific for 5.8S species extended into ITS2; (lower panel) hybridization with probe specific for the mature 5.8S rRNA. (B) Hybridization with a probe specific for the 5′ETS species. Probes used are indicated in parentheses to the left of each panel. The long and short forms of 5.8S rRNA are clearly resolved.

FIG. 4.

Rrp47p is required for 3′ processing of snoRNAs. Northern blot analyses of snoRNA species were resolved in 6% (A and F) or 8% (B to E) acrylamide-urea gels. RNA was recovered from wild-type and mutant strains as in Fig. 2. (A) Hybridization with an snR8-specific probe; (B) hybridization with an snR13-specific probe (a truncated form [snR13_T] detected in the exosome mutants is indicated); (C) hybridization with a probe specific for 3′-extended U14 species; (D) hybridization with a probe specific for 3′-extended U18 species; (E) hybridization with an snR38-specific probe; (F) hybridization with an snR44-specific probe. Discrete, 3′-extended snoRNA precursors detected in the wild-type strain (snR8-3′, U14-3′, U18-3′, snR38-3′ and snR44-3′), and the snoRNAs with short, 3-nt extensions that are specifically detected in the rrp47-Δ and rrp6-Δ mutants (snR13+3, U14+3, U18+3, and snR38+3) are indicated. The lower panels show appropriate exposures of the hybridized blots to reveal clearly the mature snoRNAs. The probes used to detect snoRNA species are indicated in brackets to the left of each panel. The electrophoretic mobilities of SCR1 (525 nt), 7S pre-rRNA (288 nt), 5.8S+30 (188 nt), 5.8S rRNA (158 nt), U14 (126 nt), and U18 (102 nt), determined by hybridization of the same filters, are indicated as size markers. The faster mobility of snR44 in the rrp4-1 mutant is due to differences in the strain background.

FIG. 5.

Analysis of U4 and U5 snRNA species in the rrp47-Δ mutant. RNA was recovered from wild-type and mutant strains as in Fig. 2 and resolved in a 6% acrylamide-urea gel. (A) Analysis of U4 snRNA. (Upper panel) Hybridization with a probe specific for 3′-extended U4 snRNA species; (center panels) hybridization with a probe complementary to the mature U4 snRNA; (lower panel) control hybridization with a probe complementary to SCR1. (B) Analysis of U5 snRNA. Hybridization was performed with a probe complementary to the mature U5 snRNA. (Upper panel) long exposure (2 days) to reveal the 3′-extended U5 snRNA precursors; (middle panel) short exposure (3 h) to reveal the relative levels of the U5_L and U5_S snRNAs; (lower panel) hybridization with a probe complementary to SCR1. The probes used are indicated in brackets on the left of each panel.

FIG. 6.

Northern blot analyses of the rrp47-Δ rrp6-Δ double mutant. RNA from rrp47-Δ (lane 1), rrp47-Δ rrp6-Δ (lane 2), rrp6-Δ (lane 3), and wild-type (lane 4) sister strains grown at 30°C were resolved through an 8% acrylamide gel, transferred, and hybridized with probes specific for mature 5.8S rRNA (A), U14 3′-extended species (B), and mature snR38 (C). The probes used are indicated in brackets to the left of each panel.

FIG. 7.

Rrp47p is not required for nuclear mRNA surveillance or cytoplasmic mRNA decay. (A) Northern blot analysis of RNA isolated from rna14-1, rna14-1 rrp6-Δ, rna4-1 rrp47-Δ, and rna14-1 GAL::rrp41 mutants during growth at 23°C (zero time points) and after transfer to 37°C for 15, 30, and 60 min. Blots were hybridized with probes specific to ACT1 and CYH2 mRNAs and to 18S rRNA as a loading control. A nonspecific RNA detected with the CYH2 probe is indicated with an asterisk. The probes used are indicated in brackets to the left of each panel. (B) Plate growth assay of the rna14-1, rna14-1 rrp47-Δ, and rna14-1 rrp6-Δ mutants on YPD medium at 25 and 36°C. Plates were incubated for 5 days. (C) Northern blot analysis of RNA from wild-type (lane 1), rrp47-Δ (lane 2), and ski7-Δ (lane 3) strains expressing the MFA2pG reporter transcript. Hybridization was performed with probe 487, complementary to the 3′ end of the poly(G) cassette within the MFA2 3′ untranslated region. The probe detects the full-length MFA2pG transcript and the poly(G)→3′ fragment (pG→3′). Shortened degradation intermediates of the poly(G)→3′ fragment detectable in the ski7-Δ mutant are indicated by a bracket on the right-hand side of the panel. The lower panels show control hybridizations of the same Northern with probes complementary to the 5.8S rRNA (oligonucleotide 017) and the SCR1 RNA (oligonucleotide 250).