Rrp17p is a eukaryotic exonuclease required for 5' end processing of Pre-60S ribosomal RNA

Abstract

Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3' end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5' end maturation. Here, we identify Rrp17p as a previously unidentified 5'-3' exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 3' end formation. Rrp17p is required for efficient exonuclease digestion of the mature 5' ends of 5.8S(S) and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.

Figure 1

Isolated Rrp17p-Associated Complexes Contain Pre-60S Ribosomal and Nuclear Peripheral Components (A) Left: Protein A (PrA) was expressed under the control of the endogenous ZPR1 promoter and affinity purified using IgG-conjugated magnetic beads (Oeffinger et al., 2007). Right: Rrp17p-associated complexes were affinity purified via the PrA tag. Proteins associated with the isolated tagged complexes were resolved by SDS-PAGE and visualized by staining with Coomassie blue. Proteins identified by mass spectrometry are listed on the right. (B) Rrp17p is a nucleolar protein. A Rrp17-GFP strain, coexpressing the nucleolar marker DsRedNop1p, was examined for localization of Rrp17p in live cells. Bar represents 10 μm.

Figure 2

Rrp17p Depletion Affects Maturation of 60S RNA Components (A) Northern analysis of high-molecular-weight RNA. RNA was extracted from wild-type and P_GAL::rrp17 strains during growth on permissive raffinose/galactose/sucrose-containing medium and after transfer to glucose medium. Pre-rRNAs are indicated schematically on the right. Rectangles represent the mature rRNA and thin lines the transcribed spacers, with the position of the probe used underlined. Probe names are indicated in parentheses on the left. (B) Northern analysis of low-molecular-weight RNA extracted and represented as indicated for (A). (C) rRNA processing pathway depicting the affects of Rrp17p depletion on synthesis of 5.8S and 25S rRNAs (bold arrows, favored pathways; dashed arrows, disrupted pathways). 5.8S rRNA synthesis has shifted from the major short to the minor long form; 26S to 25S rRNA conversion is carried out inefficiently. (D) Primer extension analysis using a primer complementary to the 5′ region of 25S rRNA. The pre-rRNA corresponding to the identified stops is indicated in parentheses on the right.

Figure 3

Rrp17p Is Needed for Efficient 5′ End Formation of 5.8S_S and 25S rRNAs (A) Northern analysis of high-molecular-weight RNA. RNA was extracted from wild-type, P_GAL::rrp17, P_MET::rat1/xrn1Δ, and P_GAL::rrp17/P_MET::rat1/xrn1Δ strains during growth on permissive synthetic dropout medium and after transfer to glucose containing medium ±5 mM methionine for the times indicated. (B) Primer extension using a primer complementary to the 5′ region of 5.8S rRNA. Pre-rRNAs are indicated schematically on the right. (C) Northern analysis of low-molecular-weight RNA extracted as described for (A). (D) Northern analysis of low-molecular-weight RNA. RNA was extracted from wild-type, P_GAL::rrp17/rai1Δ, rai1Δ, and P_MET::rat1/xrn1Δ/rai1Δ strains during growth on permissive synthetic dropout medium and after transfer to glucose synthetic dropout medium ±5 mM methionine for the times indicated. ^∗ denotes putative endonucleolytic cleavage product.

Figure 4

Analysis of RNA Binding and Exonuclease Function of Rrp17p In Vitro (A) Rrp17p binds to pre-rRNA in vitro. Gel mobility shift assay performed with an in vitro transcribed αP³²-UTP-labeled pre-rRNA fragment and recombinant (His)₁₀-Rrp17p. Lanes 1–6, pre-rRNA was incubated with 0–200 nmol Rrp17p as indicated; lane 7, pre-rRNA was incubated with 100 nmol (His)₁₀. Complexes were resolved by electrophoresis in native 6% acrylamide/bisacrylamide. (B) Degradation of in vitro transcribed 5′γP³²-ATP or 3′αP³²-pCp end-labeled mRNA by Rrp17p. Nucleic acids were resolved on a 20% acrylamide/urea gels. For the times indicated, 0.5 pmol RNA was incubated with 50 nM Rrp17p at RT. (C) Degradation of RNA substrates containing different 5′ end modification by Rrp17p. The results of four experiments were averaged for each substrate and plotted against each other to determine degradation efficiency of Rrp17p in the presence of 5′ end modifications (error bars ±3%).

Figure 5

Rrp17p Contains a Highly Conserved Domain that Is Responsible for Exonuclease Activity (A) Alignment of Rrp17p with fungal and higher eukaryotic homologs using a ClustalW alignment algorithm (Altschul et al., 1997). Individual residues with more than 80% identity across the whole alignment are shown in red and as capital letters on the consensus line. Numbers in parentheses indicate the residue numbers of aligned sequences. (B) Schematic overview of point mutations introduced in the conserved domain of Rrp17p. (C) Top: Gel mobility shift assay performed with an in vitro transcribed pre-rRNA fragment. αP³²-UTP pre-rRNA was incubated alone (lane 1) or with 50 nmol Rrp17p WT and mutant proteins for 30 min. Bottom: Degradation of in vitro transcribed 5′γP³²-ATP mRNA by Rrp17p. RNA was incubated with 50 nM of either Rrp17p or mutant proteins for 10 min at RT. (D) As a control, an RRP17 shuffle strain (rrp17::KanMX6/pURA3-RRP17) was transformed with an empty TRP1 vector (pRS414). Phenotypes of RRP17 point and truncation mutants were selected against by plating on 5-FOA plates, which causes the subsequent loss of pURA3-RRP17. Growth phenotypes were determined after growing transformants at 23°C, 30°C, and 37°C for 4 days. (E) Complementation of the RRP17 deletion by wild-type RRP17 and NOL12 (hRRP17) was assessed by transforming the RRP17 shuffle strain with constructs carrying wild-type RRP17 or NOL12. The transformants were grown for 4 days at 23°C on medium containing 5-FOA.

Figure 6

Export of Pre-60S Subunits Is Affected by Depletion of both Rrp17p and Rat1p Localization of pre-60S (ITS2-1) and pre-40S (ITS1) ribosomal subunits in wild-type, P_GAL::rrp17, P_MET::rat1/xrn1Δ, and P_GAL::rrp17/P_MET::rat1/xrn1Δ cells. Cells were grown in permissive medium to mid log phase and then shifted to restrictive medium for up to 12 hr before being fixed and mounted with DAPI (blue) to stain the nuclei. Nucleolar distribution of 5′ITS2-1 (green) and ITS1 (red) was determined in permissive and restrictive conditions. Bars represent 10 μm.

Figure 7

Conditions that Disrupt Nuclear Transport Lead to a Redistribution of Late Preribosomal Processing Factors to the Nuclear Periphery (A–J) Nuclear transport was disrupted using a metabolic poison cocktail (Shulga et al., 1996). Localization of GFP-tagged Rrp17p (A and B), Noc1p (C and D), Noc3p (E and F), Rrp12p (G and H), and Dbp5p (I and J) was determined in vivo prior to poison treatment and after 30 min in metabolic poison. Bars represent 10 μm.