Coordinated Ribosomal ITS2 RNA Processing by the Las1 Complex Integrating Endonuclease, Polynucleotide Kinase, and Exonuclease Activities  Lisa Gasse, Dirk Flemming, Ed Hurt  Molecular Cell  Volume 60, Issue 5, Pages 808-815 (December 2015) DOI: 10.1016/j.molcel.2015.10.021 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 60, 808-815DOI: (10.1016/j.molcel.2015.10.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 In Vitro ITS2 Processing in Isolated Pre-60S Particles Catalyzed by the Purified Las1 Complex (A) ITS2 processing scheme beginning with 27SB pre-rRNA, the last precursor containing unprocessed ITS2. For full processing scheme, see Figure S1A. (B) Affinity purification of TAP-tagged Las1, Erb1, and Rsa4 from yeast lysates. Eluates were analyzed by SDS-PAGE and Coomassie staining and subsequently tested for in vitro C2 processing. For Las1-TAP, bands indicated on the right were identified by mass spectrometry. The upper star (∗) marks a Las1 degradation band, the lower star IgG. (C and D) In vitro C2 cleavage catalyzed by the purified Las1-TAP complex using TAP-Erb1 or TAP-Rsa4 particles as a substrate, in the absence or presence of ATP. RNA was analyzed by primer extension to detect 27S, 26S, 25S′ pre-rRNA, and 25S rRNA (C) and northern blotting to detect 7S pre-rRNA, 5.8S, and 5S rRNA (D). (E) High-salt extraction of Las1-TAP yields a Las1-Grc3 heterodimer active in C2 cleavage. Las1-TAP affinity purified in low-salt (100 mM) or high-salt (600 mM NaCl) buffer, as well as TAP-Erb1, was analyzed by SDS-PAGE and Coomassie staining. (F and G) In vitro C2 cleavage catalyzed by the purified high-salt or low-salt Las1-TAP using TAP-Erb1 as rRNA substrate, in the absence or presence of ATP. RNA was analyzed by primer extension (F) and northern blotting (G). The white line indicates a superfluous lane that was cut from the picture in both (F) and (G). See also Figure S1. Molecular Cell 2015 60, 808-815DOI: (10.1016/j.molcel.2015.10.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Las1 Complex Mutants Mapping in Catalytic Residues Are Non-viable and Defective in ITS2 Processing (A) Affinity purification of TAP-Rsa4/Las1-AID particle and of Las1-Grc3 heterodimers, split-tag affinity purified via wild-type or mutant Las1-TEV-ProtA and Flag-Grc3. Eluates were analyzed by SDS-PAGE and Coomassie staining and subsequently tested for in vitro C2 processing. (B and C) In vitro C2 cleavage catalyzed by the purified wild-type and indicated Las1 Las1H134A or R129A mutant complexes using TAP-Rsa4/Las1-AID particle as rRNA substrate. RNA was analyzed by primer extension (B) and northern blotting (C). (D) Cell viability of las1 catalytic mutants. LAS1 shuffle strain transformed with plasmids encoding the indicated wild-type and mutant Las1 proteins under endogenous LAS1 promoter control. Transformants were analyzed for growth by spotting in 1/10 dilution series on SDC-Leu (plating control) or on SDC + FOA (5-fluoro-orotic acid) plates (for complementation). They were grown for 2 days at 30°C. (E) Affinity purification of TAP-Rsa4/Las1-AID particle and split-tag affinity purification of four subunit (4su) Las1-Grc3-Rat1-Rai1 complexes via wild-type or mutant Grc3 or Rat1. Eluates were analyzed by SDS-PAGE/Coomassie staining and subsequently tested for in vitro ITS2 processing and mode of pre-rRNA cleavage. The different running behavior of Grc3 in lanes 2–4 versus 5 and 6 is due to using Flag-tagged Grc3 versus untagged Grc3. (F) In vitro ITS2 processing catalyzed by purified Las1 complexes, harboring intact or mutant Grc3 and Rat1 subunits. RNA was analyzed by primer extension. For northern blotting, see Figure S2F. Note that 27S pre-rRNA cleavage was less complete in the case of wild-type Las1 heterotetramer isolated via Rat1 in the second purification step in the absence of ATP, but complete in the presence of ATP. See also Figure S2. Molecular Cell 2015 60, 808-815DOI: (10.1016/j.molcel.2015.10.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Las1 Cleaves 27SB Pre-rRNA at Site C2, Leaving a 2′,3′ Cyclic Phosphate at the 3′ End of 7S and Free Hydroxyl Group at the 5′ End of 26S Pre-rRNA Phosphorylated by Grc3 (A) Grc3-dependent phosphorylation of the 5′-OH end of 26S generated by Las1-dependent C2 cleavage. Extracted RNA was analyzed by 6% TBE-Urea PAGE followed by autoradiography. In lanes 6–10, 26S pre-rRNA is only labeled with [γ-32P]ATP if wild-type Grc3 is present (lane 8), but not with mutant Grc3 (lane 9 and 10), although the cleaved fragments are present in all samples containing wild-type Las1 (lanes 2–5), which can be labeled with T4 PNK. (B) Las1 endo-cleavage generates a 2′,3′ cyclic phosphate at the 3′ end of 7S pre-rRNA. Extracted RNA was analyzed by 6% TBE-Urea PAGE followed by autoradiography. Lanes 1 and 2 depict the input of all conditions visualized by labeling the 5′ end with T4 PNK. The 3′ end of 7S pre-rRNA is only accessible for 3′ labeling with T4 RNA ligase after T4 PNK treatment (2′,3′ cyclic phosphatase activity) (lane 10), but not after rSAP treatment (3′ phosphatase activity) (lane 6). (C) Scheme of ITS2 rRNA processing by the ITS2 processome (Las1-Grc3-Rat1-Rai1). Las1 cleaves 27SB pre-RNA into 7S (2′,3′ cyclic phosphate at the 3′ end) and 26S (5′-hydroxy at the 5′ end) pre-rRNA. 26S pre-rRNA is 5′ phosphorylated by Grc3 using ATP and further processed by Rat1-Rai1. See also Figure S3. Molecular Cell 2015 60, 808-815DOI: (10.1016/j.molcel.2015.10.021) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Structural and Biochemical Characterization of the Las1-Grc3-Rat1-Rai1 Complex (A) Y2H interaction between Las1 and Grc3. Y2H plasmids expressing the indicated GAL-BD (GAL4 DNA binding domain) and GAL-AD (GAL4 activation domain) constructs with the indicated amino acid boundaries were transformed into a yeast reporter strain and grown on the indicated plates. Growth on SDC-Ade reveals a strong Y2H interaction. p53-SV40 interaction served as positive control. (B) Multiple sequence alignment of the Las1 C-terminal part, which interacts with Grc3, derived from S. cerevisiae, C. thermophilum, D. rerio, and H. sapiens orthologs using ClustalW2 and Jalview. Multiple sequence alignment of the corresponding full-length Las1 sequences is shown in Figure S4A. (C) Assembly of a stable Grc3-Rat1-Rai1 core complex in the absence of Las1. Split-tag affinity purification of ProtA-TEV-Grc3 in the first and Rat1-Flag in the second step from yeast lysates in the presence (lane 1) and absence (lane 2) of Las1. (D) Electron microscopy of the purified Las1-Grc3-Rat1-Rai1 complex. Upper part, selected class averages of the negatively stained unfixed Las1 complex, split-tag affinity purified via Las1-TEV-ProtA and Rat1-Flag. Lower part, typical class average of the Las1-complex indicating its dimensions, compared to the crystal structure of the S. pombe Rat1-Rai1 heterodimer (Xiang et al., 2009) (red and blue, PDB: 3FQD) and of a Grc3 homology model based on the Clp1 crystal structure (Noble et al., 2007) (green; residues 75–557; obtained by Phyre prediction). For both, the Rat1-Rai1 and Grc3 model density maps were calculated at 30 Å resolution and are shown at the same scale as the EM class average of the Las1 complex. See also Figures S4B and S4C. Molecular Cell 2015 60, 808-815DOI: (10.1016/j.molcel.2015.10.021) Copyright © 2015 Elsevier Inc. Terms and Conditions