Suppressor mutations in Rpf2-Rrs1 or Rpl5 bypass the Cgr1 function for pre-ribosomal 5S RNP-rotation

Abstract

During eukaryotic 60S biogenesis, the 5S RNP requires a large rotational movement to achieve its mature position. Cryo-EM of the Rix1-Rea1 pre-60S particle has revealed the post-rotation stage, in which a gently undulating α-helix corresponding to Cgr1 becomes wedged between Rsa4 and the relocated 5S RNP, but the purpose of this insertion was unknown. Here, we show that cgr1 deletion in yeast causes a slow-growth phenotype and reversion of the pre-60S particle to the pre-rotation stage. However, spontaneous extragenic suppressors could be isolated, which restore growth and pre-60S biogenesis in the absence of Cgr1. Whole-genome sequencing reveals that the suppressor mutations map in the Rpf2-Rrs1 module and Rpl5, which together stabilize the unrotated stage of the 5S RNP. Thus, mutations in factors stabilizing the pre-rotation stage facilitate 5S RNP relocation upon deletion of Cgr1, but Cgr1 itself could stabilize the post-rotation stage.

Fig. 1

The short α-helical protein Cgr1 is wedged on nucleoplasmic pre-60S particles close to the rotated 5S RNP. a Cryo-EM position of Cgr1 wedged between the β-propeller domain of Rsa4 and the rotated 5S RNP on the Rix1–Rea1 pre-ribosomal particle (PDB: 5jcs,). The 5S rRNA (blue) and H38 of the 25S rRNA (orange) are shown as a surface models, Cgr1 (red), Rsa4 (purple), Rea1 (cyan), Rix1-complex (light blue), Rpl5 (dark blue), and other ribosomal proteins (RPL, light blue) are depicted. b Multiple sequence alignment of Cgr1 orthologues from different fungal species: Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Kluyveromyces lactis (Kl), Yarrowia lipolytica (Yl), Chaetomium thermophilum (Ct) and Neurospora crassa (Nc); for the sequence alignment with higher eukaryotic orthologues including human Cgr1, see Supplementary Fig. 9. Two mutant constructs, Cgr1ΔN51 and Cgr1RRR > AAA, used for genetic interaction studies, are indicated above the alignment. c Subcellular distribution in yeast cells of GFP-tagged Cgr1 and RFP-Nop1 was monitored by fluorescence microscopy. The localization of GFP–Cgr1 is distributed over the nucleus, with the tendency to show a slightly stronger signal in the nucleolus. Scale bar is 5 µm. d, e Cgr1 is co-enriched on intermediate pre-60S particles typically found in the nucleus. d Cgr1 tagged either N-terminally (TAP–Flag) or C-terminally (FTpA) were isolated from yeast lysates in two affinity-purification steps. The final Flag eluates were analysed by SDS-PAGE followed by Coomassie staining. The bands identified by mass spectrometry are indicated. e Different pre-60S particles affinity purified via bait proteins Ssf1–FTpA (early nucleolar), Nsa1–FTpA (early nucleolar), Nog2–FTpA (intermediate nucleoplasmic), Rix1–FTpA (intermediate nucleoplasmic), Arx1–FTpA (intermediate nucleoplasmic to late cytoplasmic) and Lsg1–FTpA (late cytoplasmic) were affinity purified from yeast strains, which expressed Cgr1 carrying 3xHA (Cgr1–3xHA). Final eluates were analysed by SDS-PAGE and Coomassie staining (upper panel) or western blotting, using the indicated antibodies detecting Nog1, Nog2, Rpl3 and Cgr1 (lower panels). M: molecular weight marker

Fig. 2

Cgr1 plays a crucial role in ribosome biogenesis of pre-60S particles. a Chromosomal CGR1 deletion in wild-type yeast strain W303 yields viable cells with an extreme slow-growth phenotype. The cgr1Δ shuffle strains transformed with empty plasmid or plasmid carrying wild-type CGR1 were shuffled on SDC + FOA plates, before representative colonies were spotted in 10-fold serial dilutions on YPD plates. They were grown at the indicated temperatures for 2 days. b Cgr1 depletion impairs 60 S subunit synthesis. Polysome-profiles of CGR1–HA–AID (i.e. Cgr1-depletion strain) were recorded for untreated or auxin-treated (for 120 min) cells. Arrows denote ribosomal half-mers, indicating a specific 60S biogenesis defect. c pre-60S export is inhibited in cells depleted of Cgr1. Subcellular localization of the 60S reporter Rpl25–GFP, the 40S reporter Rps3–GFP and the nucleolar marker RFP–Nop1 was analysed in untreated or auxin-treated (for 120 min) CGR1-HA-AID cells. Arrows indicate nuclear accumulation of Rpl25–GFP. Scale bar is 5 µm. d Depletion of Cgr1 shifts Arx1 pre-60S particles to the early pool typical for the unrotated 5S RNP. Arx1–FTpA particles were affinity purified from untreated or auxin-treated (for 120 min) CGR1–HA–AID cells expressing a chromosomally integrated RPF2–3xHA variant. Lysates serving as input for the purifications and final eluates were analysed by SDS-PAGE and Coomassie staining. Indicated bands were identified by mass spectrometry (left panel, asterisk indicates Rpf2-3xHA) and western blotting based on specific antibodies (right panel). Rpf2 carries a 3xHA tag, whereas only one HA epitope is fused to Cgr1, explaining the different signal intensities of the HA western blots. e Synthetic lethal relationship between cgr1 mutant alleles and distinct pre-60S assembly factors. Double-shuffle strains of cgr1Δ in combination with nsa2Δ, rix1Δ, nug1Δ and nop7Δ, respectively, were co-transformed with indicated plasmid-based wild type and mutant constructs. Transformants were spotted in 10-fold serial dilutions and growth on SDC-Leu-Trp (SDC) and SDC + FOA plates at 30 °C was monitored after 2 and 6 days, respectively. The cgr1RRR > AAA and cgr1ΔN51 mutants are shown in Fig. 1b and Supplementary Fig. 3. Published mutant alleles nsa2-1, rix1-1, nug1-1 and nop7-1 are listed in Supplementary Table 3

Fig. 3

Suppressor mutations in RPF2, RRS1 and RPL5 bypass the requirement for CGR1. a Dot spot growth analyses of the cgr1Δ strain, harbouring plasmid-borne CGR1 (left panel) or empty plasmid (middle and right panels), incubated on YPD plates at 30 °C for 3 days. The dot spot on the right, but not the middle, exhibits faster-growing colonies, which are suppressors of cgr1Δ. b cgr1Δ (cgr1::natNT2) suppressor strain was crossed with a cgr1Δ (cgr1::HIS3MX6) strain containing CGR1 on a URA3 plasmid (pRS316-CGR1). After sporulation and tetrad dissection (upper panel shows a representative tetrad), the four haploid spores were tested for growth in the absence of pRS316-CGR1 on SDC + FOA plates, for the presence of pRS316-CGR1 on SDC-Ura, and for the presence of the CGR1 gene disruption markers on SDC-His and YPD + clonNat. Cells were spotted in 10-fold serial dilutions and incubated at 30 °C for 2 days (lower panel). c–f Suppressor mutations are located in genes encoding RPF2, RRS1 and RPL5. c Wild type and different cgr1Δ suppressor strains (suppressor #1, #3 and #19) were transformed with plasmids expressing RPF2, RRS1, RPL5 or RPL11 under the control of the galactose-inducible GAL1-10 promoter. Representative transformants were spotted in 10-fold serial dilutions on SDC plates containing glucose (GAL repression) and galactose (GAL induction) and growth was assessed after incubation at 30 °C for 2 and 3 days, respectively. d, e Double-shuffle strains of cgr1Δ ( + pURA3-CGR1) combined with rpf2Δ ( + pURA3-RPF2), rrs1Δ ( + pURA3-RRS1) and rpl5Δ ( + pURA3-RPL5), respectively, were transformed with plasmids harbouring the suppressor allele or the respective wild-type gene combined with plasmids harbouring wild-type CGR1 or empty plasmid. Transformants were spotted in 10-fold serial dilutions on SDC + FOA plates (d) and after plasmid shuffling on YPD plates (e). Growth was analysed after incubation for 2 days at the indicated temperatures. f Multiple sequence alignment of Rpf2, Rrs1 and Rpl5 orthologues from Saccharomyces cerevisiae (S.c.), Chaetomium thermophilum (C.t.), Neurospora crassa (N.c.), Kluyveromyces lactis (K.l.), Yarrowia lipolytica (Y.l.), Caenorhabditis elegans (C.e.), Mus musculus (M.m.), Homo sapiens (H.s.), Pyrococcus horikoshii (P.h.) and Halobacterium hubeiense (H.h.). The respective suppressor alleles analysed in d and e are indicated

Fig. 4

cgr1Δ suppressor mutations in RPF2, RRS1 and RPL5 destabilize the unrotated 5S RNP on the pre-60S particle. a Overview of biogenesis factors Rpf2 (orange), Rrs1 (green), Rsa4 (purple), Cgr1 (red) and the ribosomal protein Rpl5 (dark blue) on the Nog2 particle (PDB: 3jct,) in the front and top view. Ribosomal proteins are shown in light blue and 5S rRNA as a dark grey surface model filtered at 6 Å resolution. b Positions of cgr1Δ null suppressor mutations (red) displayed with surface models of the 5S rRNA (dark grey) and helices H83 to H87 (nucleotides 2650–2754) of the 25S rRNA (light grey). The KKR-loop of Rpf2 is highlighted in cyan. ESL2 and ESL3 mark the eukaryotic-specific loops 2 and 3 of Rpl5, NTH marks the N-terminal helix of Rpf2

Fig. 5

Suppressor mutations in Rpf2, Rrs1 and Rpl5 rescue the 60S biogenesis defect in Cgr1-depleted cells. a Nuclear pre-60S export is restored in suppressor mutants after Cgr1 depletion. Subcellular location of Rpl25–GFP and RFP–Nop1 (nucleolar marker) was examined in CGR1–HA–AID cells expressing either wild-type RPF2, RRS1 and RPL5 or the respective mutant alleles rpf2V203F, rrs1E102D and rpl5I190F, after incubation with auxin for 120 min. Scale bar is 5 µm. b Biochemical maturation of Arx1 pre-60S particles is restored in cgr1Δ suppressor mutants. Arx1–FTpA particles were affinity purified from CGR1–HA–AID cells expressing either wild-type RPF2, RRS1 or RPL5, or the indicated suppressor mutants before and after treatment with auxin for 120 min. Final eluates were analysed by SDS-PAGE and Coomassie staining (indicated bands were identified by mass spectrometry; upper panels) or by western blotting using the antibodies shown on the left (lower panels). The area of the Coomassie-stained SDS-polyacrylamide gel to which Rpf2 migrates is enlarged on the right to better reveal how the intensity of co-enriched Rpf2 changes, depending on Cgr1 depletion in the various suppressor mutants

Fig. 6

Cryo-EM reveals inhibition of 5S RNP rotation in cgr1Δ cells but restoration in rrs1E102D suppressor mutant. a–c 3D cryo-EM reconstructions of pre-60S particles affinity purified via the Arx1 bait protein from the indicated yeast strains. a CGR1 control strain: rrs1Δ [YCplac111–RRS1] Arx1–FTpA Cgr1–HA–AID; -auxin. b cgr1Δ depleted: rrs1Δ [YCplac111–RRS1] Arx1–FTpA Cgr1–HA–AID; + auxin (2 h). c cgr1Δ depleted in the presence of the rrs1E102D suppressor: rrs1Δ [YCplac111–rrs1E102D] Arx1–FTpA Cgr1–HA–AID + auxin (2 h). For each obtained class of the respective data set, the rotation state of the 5S is indicated by a fit model of 5S rRNA taken from PDB: 3jct (green: pre-rotation,) or PDB: 5jcs (blue: post-rotation,). Also, the presence or absence of the ITS2-harbouring foot structure is indicated