Fig 1.
Extragenic suppressors of bud23Δ reveal an interaction network that connects the 3’ basal subdomain to the U3-18S heteroduplexes.
(A) A secondary structure map of the 18S rRNA that indicates the position of the 3’ basal subdomain (dark gray) and the central pseudoknot (CPK; deep olive). (B) The position of the 3’ basal subdomain (dark gray) within the context of the SSU Processome structure (light gray). (C) Factors harboring mutations that suppress bud23Δ and are resolved in the SSU Processome (PDB 5WLC) cluster around the 3’ basal subdomain and the U3-Box A’-18S heteroduplex that Dhr1 unwinds. Shown are: Bms1 (forest green), Imp4 (blue), Rps28 (cyan), Utp2 (orange), Utp14 (brown), U3 (deep purple), 18S rRNA (deep olive), 3’ basal subdomain (dark gray). (D) Zoomed view of factors in C showing contacts amongst each other, the 3’ basal subdomain, and the U3-Box A’-18S heteroduplex. N- and C-terminal domains, NTD and CTD, respectively. The U3-18S heteroduplexes are shown as U3 Box A and U3 Box A’. Guanosine 1575 (G1575, red) is shown as a marker for the binding site of Bud23. (E) Tabulation of the number of unique mutations found in each extragenic suppressor of bud23Δ. Newly identified mutations (novel) and previously identified (known) [30,45–47]. The complete list of these mutations is available in S1 Table. (F) Summary of the genetic and physical interactions amongst the suppressors of bud23Δ. Factors are indicated as nodes; genetic and physical interactions are shown as dashed and solid edges, respectively.
Fig 2.
The mutated residues in Imp4 and Rps28A primarily map to their interfaces with the 3’ basal subdomain.
(A) Point mutations within imp4 and rps28a suppressed the growth defect of bud23Δ as shown by 10-fold serial dilutions of wild-type cells (BY4741), bud23Δ (AJY2676), and bud23Δ-suppressed cells spotted on YPD media and grown for two days at 30°C. (B) Rps28 and the Brix domain of Imp4 interact with the 3’ basal subdomain RNA, while the NTD of Imp4 makes contacts with its Brix domain and the U3-18S heteroduplexes. G1575, the binding site of Bud23, is shown for reference. The regions where the mutated residues map are indicated by magenta and green dashed boxes for the rRNA interaction and the NTD interaction, respectively. Factors are colored the same as in Fig 1. (C) Residues mutated in Rps28 and Imp4 Brix domain map to interaction interfaces with the 3’ basal subdomain RNA (magenta sticks). (D) Several residues mutated in Imp4 map to an intramolecular interaction between the Brix and NTD of Imp4 (green sticks). (E) Suppressing mutations in imp4 and rps28a partially restored A2 processing and 18S rRNA levels in bud23Δ cells. RNA processing intermediates were detected by Northern blotting on RNAs extracted from wild-type (WT), bud23Δ, and bud23Δ-suppressed cells cultured to exponential phase at 30°C in liquid YPD. P32-radiolabeled probes (Table 3) hybridized to the indicated regions. The 25S and 18S rRNAs were detected by methylene blue staining of the RNAs prior to oligonucleotide hybridization. (F) The imp4 and rps28a mutations partially restored 40S biogenesis as shown by polysome profiles after separation of extracts on sucrose density gradients from wild-type, bud23Δ, and bud23Δ-suppressed cells cultured to exponential phase at 30°C in liquid YPD media.
Fig 3.
The Utp2 mutants lose interaction with Imp4.
(A) Spontaneous point mutations within utp2 partially suppressed the growth defect of bud23Δ as shown by 10-fold serial dilutions of wild-type cells (BY4741), bud23Δ (AJY2676), and bud23Δ-suppressed cells spotted on YPD media and grown for two days at 30°C. (B) Additional point mutations in UTP2, generated by error-prone PCR, also suppressed the growth defect of bud23Δ (left) and complemented loss of UTP2 (right) as shown by 10-fold serial dilutions of bud23Δ (AJY2676) and PGAL10-UTP2 (AJY4175) cells containing either empty vector (pRS416), or vectors encoding the indicated alleles of UTP2 spotted on SD-Ura media containing glucose and grown for two days at 30°C. (C) Several of the amino acid substitutions in Utp2 map to residues (green sticks) located within its NTD (orange) that interacts with the Brix domain of Imp4 (blue) adjacent to the 3’ basal subdomain RNA (gray). (D) Left: Yeast two-hybrid interaction assay between Imp4 and wild-type (WT) or mutant Utp2. Strains carrying the indicated constructs were patched onto SD-Leu-Trp- (L-W-) and SD-Leu-Trp-His- (L-W-H-) media supplemented with 2 mM 3-Amino-1,2,4-triazole (3AT) (AD, Gal4 activation domain; BD, Gal4 DNA binding domain). Right: Western blot analysis of the wild-type and mutant Utp2-AD-HA proteins using equivalent amounts of total protein extracts. Glucose-6-phosphate dehydrogenase (G6PDH) was used as a loading control. (E) Top: F58 of Utp2 (green sticks) fits into a hydrophobic pocket in the Brix domain of Imp4 (surface representation). Bottom: The bud23Δ-suppressing mutations V170F and P252L of Imp4 (magenta sticks) line this pocket. Imp4 and Utp2 are colored blue and orange, respectively. (F) Top: Yeast two-hybrid interaction assay between Utp2 and wild-type or mutant Imp4. Strains carrying the indicated constructs were patched onto L-W- and L-W-H- media supplemented with 6 mM 3AT. Bottom: Western blot analysis of the wild-type and mutant BD-myc-Imp4 proteins in equivalent amounts of total protein extract is shown. G6PDH was used as a loading control.
Fig 4.
The mutated residues in the GTPase Bms1 that suppress bud23Δ are poised to modulate its conformational state.
(A) Spontaneous point mutations within BMS1 suppressed the growth defect of bud23Δ as shown by 10-fold serial dilutions of wild-type cells (BY4741), bud23Δ (AJY2676), and bud23Δ cells carrying the indicated bms1 mutations spotted on YPD media and grown for two days at 30°C. (B) The bms1 mutations partially restored A2 processing and 18S rRNA production in bud23Δ cells as shown by Northern blotting of RNAs extracted from wild-type, bud23Δ, and bud23Δ-suppressed cells as described in Fig 2E. (C) The bms1 mutations partially restored 40S biogenesis as shown by the analysis of the polysome profiles from the indicated strains as described in Fig 2F. (D) Top: Primary structure of Bms1 with domains (in different shades of green), interacting regions and bud23Δ suppressing mutations annotated; regions not resolved in SSU Processome structures are indicated in light gray. Bottom: The partial structure of Bms1 (from PDB 5WLC) in the context of the SSU Processome is shown. Domains IV and V extend from its GTPase core (domains I–III) to contact the RNAs of the 3’ basal subdomain (gray) and the U3-18S heteroduplexes (pink/gold), respectively. (E) At the 3’ basal subdomain, Domain IV of Bms1 also contacts the CTDs of Utp2 (orange) and Imp4 (blue). (F) The mutated residues D124, D843, A903, and S1020 in Bms1 map to inter-domain contacts with the unstructured strand of domain V that connects it to domain IV.
Fig 5.
Most of the DHR1 mutations map to surface residues of its RecA domains.
(A) A cartoon of the primary structure of Dhr1 is shown. The domains of Dhr1 are annotated by color: NTD, N-terminal domain (light gray); RecA1/2, Recombination protein A1/2 (blue/green); WH, winged-helix (yellow); HB, helical bundle (orange); OB, oligonucleotide-binding fold (brown); CTD, C-terminal domain (light red). Unstructured regions are colored as light gray. Mutations reported here (novel) and previously (known) [46] are indicated as black and magenta, respectively. Numbering indicates residue numbering of yeast Dhr1. (B) The structure of yeast Dhr1 (PDB 6H57) with relevant features colored as described in panel A. Catalytic residues involved in ATP hydrolysis are denoted as red sticks for reference. (C) The majority of the mutated residues map to the surfaces of the RecA domains. Mutated residues are shown as black and magenta sticks as described for panel A. The residues that were used to test loss-of-interaction with Utp14 in panel D are labeled. (D) Top: Yeast two-hybrid interaction data between AD-HA-Utp14 and wild-type (WT) or mutant BD-myc-Dhr1 are shown. Strains carrying the indicated constructs were patched onto SD-Leu-Trp- (L-W-) and SD-Leu-Trp-His- (L-W-H-) media supplemented with 10 mM 3-Amino-1,2,4-triazole (3AT) (AD-HA, GAL4AD-HA; BD-myc, GAL4BD-myc). Bottom: Western blot analysis of the wild-type and mutant BD-myc-Dhr1 proteins using equivalent amounts of total protein extracts is shown. Glucose-6-phosphate dehydrogenase (G6PDH) was used as a loading control.
Fig 6.
Imp4 and Enp1 accumulate with pre-40S upon Bud23 depletion.
(A) The genomic fusion of an auxin-inducible degron (AID) to the C-terminus of Bud23 rendered cells sensitive to auxin, with a growth defect comparable to bud23Δ. 10-fold serial dilutions of wild-type (AJY2665), BUD23-AID (AJY4395), and bud23Δ (AJY3156) cells were spotted on YPD media with and without 0.5 mM auxin and grown for two days at 30°C. (B) Western blot of time-course of depletion of Bud23-AID, using equivalent amounts of total protein extract from AJY2665 or AJY4395 cells cultured to exponential phase then harvested prior to or after the addition of 0.5 mM auxin for the indicated time (WT; wild-type). G6PDH was used as a loading control. (C) The sucrose density gradient sedimentation of Enp1-TAP, Imp4, and Rps24 in the presence (upper panel) or absence (lower panel) of Bud23. Extracts were prepared from + Bud23 (AJY2665) and—Bud23 (AJY4395) cells treated with 0.5 mM auxin for two hours and separated on sucrose density gradients prior to fractionation. Proteins from each fraction were precipitated and subjected to Western blot analysis.
Fig 7.
Composition of 40S precursors purified in the absence of Bud23.
(A) Coomassie-stained gel of proteins that co-purified with Enp1-TAP in the presence (+) or absence (-) of Bud23. Pre-ribosomal particles were enriched by overlaying eluate onto sucrose cushions followed by ultracentrifugation. Individual species that showed clear enrichment or depletion were excised and identified by mass spectrometry and are indicated in blue or black text, respectively. The asterisks (*) denote proteins that also appeared in the analysis described in S9 Fig. (B) The rRNA processing intermediates and U3 snoRNA that co-purified with Enp1-TAP in the presence (+) or absence (-) of Bud23 were detected by Northern blotting using the indicated probes. Oligonucleotides are listed in Table 3.
Fig 8.
Model for when Bud23 functions during SSU Processome progression.
(A) The complete SSU Processome is decorated with assembly factors (AFs; light blue) and some ribosomal proteins (RPs; teal) that establish the rRNA architecture. During the transition to the Dis-C complex Dhr1 is recruited and most SSU Processome AFs are released. The Dis-C complex harbors 15 AFs including Rps28 (cyan), Imp4 (blue), Utp2 (orange), Bms1 (green), Utp14 (brown) and Dhr1 (pink). The incorporation of Bud23-Trm112 (light yellow and light blue, respectively) promotes the release of the residual SSU Processome AFs, resulting in the final disassembly of the SSU Processome to generate an early pre-40S complex. (B) In the complete SSU Processome, the rRNA of the 5’ (blue), central (yellow), 3’ major (magenta), and 3’ minor (green) domains are splayed apart by the U3 snoRNA (gray) and the 5’ ETS (white). These domains become partially compacted and the 5’ ETS is released during the transition to the Dis-C complex. The release of the U3 snoRNA and the further rotation of the 3’ major and 3’ minor domains produces the early pre-40S. The yeast SSU Processome (left) is a composite PDBs 5WLC and 5WYK, the Dis-C complex (middle) is from PDB 6ZQG, and the early pre-40S (right) is from humans (PDB 6G4W). Images were generated in UCSF ChimeraX v0.93 [88].
Table 1.
Yeast strains used in this study.
Table 2.
Plasmids used in this study.
Table 3.
Oligonucleotide probes used for Northern blotting.