Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Abstract

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.

Keywords: Brix proteins; Chaetomium thermophilum; UTP-A; UTP-B; interaction map; ribosome biogenesis.

Figure 1

From gene identification to structure determination of ribosome biogenesis factors. (A) Scheme depicts the workflow from in silico ORF annotation to cloning, expression, determination of the interactome, biochemical characterization and structural analysis by cryo EM or crystallography. (B) Statistics of the structural approach. From 90 targets that were tested for expression in E. coli (column A), 77 were well expressed (column B) and 52 were soluble (column C), which went into large‐scale purification. Subsequently, 40 proteins were suitable for crystallization trials (column D), of which 24 yielded crystals (column E), and finally allowed determination of 14 structures (column F). (C,D) Crystal structure of the phosphatase domain (PD) of ctTif6 (C, pdb: 5M3Q) and ctYvh1 (D, pdb: 5M43) both colored from N‐ to C‐terminus (blue to red). The overall structure of ctTif6 is highly similar to that of scTif6 (RMSD 0.67 Å over 216 residues), with an internal fivefold symmetry. The phosphatase domain of ctYvh1 belongs to the family of dual‐specificity phosphatases, able to hydrolyze phosphate from phosphorylated serine/threonine as well as tyrosine residues.

Figure 2

Illustration of the screening procedure for Y2H interactions. (A) Scheme of the experimental setup of the Y2H screen. Yeast strain PJ69‐4 MATa was transformed with 181 different Prey plasmids pGADT7 and a mix of five transformants was transferred to one position within two 96 deep well plates, representing the yeast‐two‐hybrid (Y2H) library. During the screening procedure, a liquid culture of the yeast strain (PJ69‐4 MATalpha) carrying the bait protein (pGBKT7) was mated against the refreshed library in a 96 deep well plate in liquid YPD medium. The next day, YPD was replaced with SDC‐Trp‐Leu medium to select for diploid cells carrying both plasmids. (B) After 2 day's incubation (30°C), the cells were spotted on SDC‐Trp‐Leu plates to control the mating efficiency, and on SDC‐Trp‐Leu‐His + 1 mM 3‐AT and SDC‐Trp‐Leu‐Ade plates to screen for medium and strong interactions. Plates were incubated at 30°C and documented after four and 7 days. Examples are shown in B. Bait proteins are listed on the left side and interacting prey proteins are indicated at the right side. (C) The strength of the Y2H interaction was ranked based on size and numbers of colonies. The total score was calculated from the mean value of 2 repetitions and the sum of the score derived from growth on SDC‐Trp‐Leu‐His11 mM 3‐AT and SDC‐Trp‐Leu‐Ade plates. The detected interactions are summarized in Supporting Information Tables S3 and S4 and can be viewed together with the primary data at http://y2h.embl.de.

Figure 3

Y2H interaction within the ctUTP‐A and ctUTP‐B complex. (A) The interaction network as derived from the large‐scale screen is depicted. An arrow from the prey protein (AD) towards the bait protein (BD) shows a positive interaction between the connected proteins. The displayed network is derived from the interactive software tool that contains the complete dataset that can be accessed at http://y2h.embl.de. (B) The Y2H interactions from the screen were confirmed by co‐transformation into yeast strain PJ69‐4 (see “Materials and Methods”). The transformants were analyzed for growth on SDC‐Trp‐Leu‐His (SDC‐HIS, weak interactions) and SDC‐Trp‐Leu‐Ade (SDC‐ADE, strong interactions) at 30°C. The growth phenotype after 2 days is shown. The upper panels show the results of the ctUTP‐A complex; the lower panels show the data for ctUTP‐B.

Figure 4

Biochemical reconstitution of the ctUTP‐A and ctUTP‐B complex. Thermophilic proteins were recombinantly co‐expressed in E. coli or S. cerevisiae and co‐purified (for details, see “Materials and Methods”). The dimeric and trimeric reconstituted complexes of ctUTP‐A (A) and ctUTP‐B (B) were analyzed by SDS‐PAGE and subsequent Coomassie blue staining. The label at the right indicates the position of the protein with its tag. A protein A tagged protein, which gets cleaved from its tag during purification is marked with an asterisk. (C) For the reconstitution of the complete ctUTP‐A (left panel) and ctUTP‐B (right panel) complex, all members were recombinantly co‐expressed in yeast and isolated using a split tag approach.

Figure 5

Structural characterization of the reconstituted ctUTP‐A and ctUTP‐B complex. The reconstituted ctUTP‐A and ctUTP‐B complexes [see Fig. 2(C)] were purified by the GraFix approach and subsequently analysed by negative stain electron microscopy. Left panel shows a micrograph, the right panel shows a gallery of the representative class averages. Scale bar indicates 10 nm.

Figure 6

Reconstitution of the Brix–BAP complexes involved in 60S biogenesis. (A) The structure of the Brix domain Rpf2 (blue) in complex with the BAP Rrs1 (red) is shown. The structure is taken from PDB entry 5BY8.69 (B–D) A Y2H analysis of the C. thermophilum Brix proteins with their BAP is shown. The upper panel shows the ctBrix domain fused to the activation domain (pGADT7) without terminal extensions [rpf1 (156–436 aa), brx1 (30–260 aa), and ssf1 (22–359 aa)] tested with the corresponding BAP fused to DNA binding domain (pGBKT7), whereas the lower panel shows the fragment of the ctBAP (pGADT7) (mak16 (134–336 aa), ebp2 (178–268 aa), and rrp15 (194–354 aa) tested with the full‐length Brix protein (pGBKT7). SDC‐Trp‐Leu‐His plates are shown after 3 days incubation at 30°C. (E–G) A binding assay (left panel) and size exclusion chromatography (SEC, right panel) was performed to verify the Y2H interactions. For the binding assay, the indicated GST proteins were immobilized on GSH beads, washed (lanes 3, 5), incubated with E. coli supernatant containing the His6 tagged partner (lane 2), washed and eluted by GSH (lane 4). As a negative control the His6 tagged proteins were incubated with GST alone (lane 6). Right panel shows SEC analysis of the affinity‐purified complexes. SDS‐PAGE shows protein composition of the peak fraction (domain boundaries are identical as in B–D except for SEC analysis of ctBrx1 (34–259 aa)—ctEbp2 (175–282 aa), and ctSsf1 (34–478 aa)—ctRrp15 (173–354 aa) complexes.