A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis

Abstract

Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.

Figure 1.

Jjj1 and Zuo1–Ssz1 have overlapping biological functions. (A) Similar domain organization of Jjj1 and Zuo1. Jjj1 contains an N-terminal J domain, two zinc fingers (ZnF), and a C-terminal R/K-rich domain. Alignment with Zuo1 defines an additional homology domain (ZHD; Fig. S2). (B) Cells deleted for both Jjj1 and either Zuo1 (top) or Ssz1 (bottom) present a synthetic growth phenotype. An equal number of cells was spotted as a 10-fold dilution series on YPD plates and incubated at 30°C for 2 d. (C) Jjj1 overexpression does not suppress the antibiotics sensitivity of Δzuo1 cells. Δzuo1 cells were transformed with an empty p426 vector, the p426-ZUO1 and p426-JJJ1. The serial dilution was performed as in B.

Figure 2.

Jjj1 and the CLIPS Zuo1, Ssz1, and SSB participate in ribosome biogenesis. (A) Joint deletion of JJJ1 and ZUO1 accumulates a 66S preribosomal particle containing the 27S rRNA precursor. (top) Lysates from WT and Δjjj1Δzuo1 cells were separated on a 7–47% sucrose gradient and the OD_{254 nm} monitored. RNA from individual fractions was analyzed on formaldehyde agarose gels and ethidium bromide staining to visualize the 25S and 18S rRNAs. (bottom) Northern blot analysis for 27S rRNA of Δjjj1Δzuo1 and WT cell lysates. (B–E) Deletion of JJJ1, ZUO1, or SSB1/2 produces aberrant polysome profiles. 20 OD_{254 nm} of yeast lysates from WT (A), Δjjj1 (B), Δzuo1 (C) or Δssb1/2 (D) were fractionated on a 7–47% sucrose gradient and the OD_{254 nm} monitored. The position of 80S ribosomes and 40S and 60S ribosomal subunits are indicated. Arrows indicate the half-mers.

Figure 3.

Distinct effects of Jjj1, Zuo1, and SSB on 60S ribosomal export and Arx1 recycling. (A) Schematic representation of ribosome biogenesis of the 60S ribosomal subunit. Nucleolar ribosomal biogenesis factors are represented in yellow, nuclear ones in green, shuttling factors, like Arx1, in blue, and cytoplasmic factors in orange. (B) Jjj1 and Zuo1, Ssz1, and SSB are involved in 60S ribosome subunit biogenesis. Total RNA from the indicated yeast cells was extracted and separated on a formaldehyde agarose gel. The 27S rRNA was detected by Northern blotting. RNA loading was controled by ethidium bromide staining to visualize the 25S and 18S rRNAs (top) and by Northern blot analysis for the 25S rRNA (bottom). (C) Effects of CLIPS deletion on the export of the 60S ribosomal subunit. The export of the 60S subunit was monitored using Rpl25-GFP as a reporter (provided by D. Roser, University of Heidelberg, Heidelberg, Germany). Cells were grown to mid-log phase at 25°C, and the in vivo localization of Rpl25-GFP was monitored by fluorescence microscopy. The nucleolar marker Sik1-RFP was used to identify the position of the nucleus. (D) Zuo1 and Jjj1 but not SSB are required for the recycling of the shuttling factor Arx1. Cells were transformed with the Arx1-GFP (provided by A. Johnson, University of Texas at Austin, Austin, TX) plasmid and were grown at 25°C to mid-log phase. The in vivo localization of Arx1-GFP was monitored by fluorescence microscopy. DIC, differential interference contrast. See Results for description of arrowheads.

Figure 4.

Jjj1 is a conserved modular protein with distinct functional domains. (A) Domain mutants of Jjj1. Jm, mutated in the canonical HPD motif of the J domain; ΔZHD, lacks the ZHD; ΔC, lacks the C-terminal K/R-rich domain. Asterisk indicates the point mutation in the HPD motif of the J domain. (B) Effect of Jjj1 mutations on the interaction with ribosomes. Yeast extracts from the indicated cells were fractionated on 7–47% sucrose gradients. The OD_{254 nm} profile (top) identifies the polysomal fractions. Individual fractions were analyzed for the presence of Jjj1 mutants and the ribosomal protein Rpl3 by SDS-PAGE and immunoblotting. Similar results are obtained by expression of Jjj1 mutants from centromeric plasmids (middle), the endogenous chromosomal copy (bottom), or high copy number plasmids (Fig. S5). (C) Cellular localization of the different Jjj1 mutants. Δjjj1 cells expressing the different mutants of Jjj1 in fusion with GFP were monitored by fluorescence microscopy. Sik1-RFP identifies the position of the nucleolus. The Jjj1-GFP fusion complements the slow growth phenotype of Δjjj1 cells (Fig. S5) and associates with ribosomes (not depicted). (D) Role of the Zuo1 C terminus and the J domain in its subcellular localization and function. Δzuo1 cells expressing the different mutants of Zuo1 in fusion with GFP were monitored by fluorescence microscopy. (E) Identification of Jjj1 domain mediating association with Rei1. (top) Domain structure of Rei1 indicating position of the three zinc fingers (yellow) and the C-terminal Rpl24-binding domain (green) are indicated (Lebreton et al., 2006). (bottom left) Yeast lysates were prepared from cells expressing HA-Jjj1 or the indicated Jjj1 domain variants together with Flag-tagged Rei1. After immunoprecipitation for Jjj1, the association with Rei1 was detected by immunoblotting with anti-Flag. Total Rei1 and Jjj1 protein was analyzed by immunoblotting against the respective tags. Asterisk indicates a background crossreacting band. (bottom right) Yeast lysates were prepared from cells carrying an endogenously tagged Jjj1 together with Flag-tagged Rei1 or Rei1 domain variants. After immunoprecipitation for Rei1, the association with Jjj1 was detected by immunoblotting with anti-Myc. Total Rei1 and Jjj1 protein was analyzed by immunoblotting. DIC, differential interference contrast; ZnF, zinc finger. See Results for description of arrowheads.

Figure 5.

A nuclear form of Jjj1 that does not bind Rei1 suffices to restore its function in ribosome biogenesis and Arx1 recycling. (A) Complementation of Δjjj1 by Jjj1 mutants. Cells expressing the Jjj1 variants were grown overnight at 30°C, and a dilution series was performed on −URA plates. (B) Jjj1-NESm is functional and complements the slow growth phenotype of the Δjjj1 cells. (C) Rescue of Δjjj1 aberrant polysome profile by Jjj1 domain mutants. Yeast lysates were fractionated on a 7–47% sucrose gradient, and the OD₂₅₄ was monitored. The gray columns indicate the 40S and the 60S peaks. The arrows indicate the presence of half-mers containing extra 48S initiation complexes. (D) Rescue of defective Arx1 recycling in Δjjj1 cells by Jjj1 domain mutants. Δjjj1 cells were transformed with the plasmids expressing the different Jjj1 mutants and the Arx1-GFP plasmid, and localization of Arx1-GFP was monitored by fluorescence microscopy. Sik1-RFP protein was used to identify the nucleus. DIC, differential interference contrast. (E and F) Overexpression of Jjj1 can suppress the slow growth phenotype of the Δssb1/2 (E) and Δzuo1 (F) cells in a domain-specific manner. Cells were transformed with either WT or mutant Jjj1, and growth was assessed by a dilution series assay. White line indicates that intervening lanes have been spliced out. See Results for description of arrowheads.

Figure 6.

Jjj1 and Zuo1 play early and distinct roles in ribosome biogenesis. (A) Association of Jjj1 and Jjj1-ΔC with nuclear and cytoplasmic (Cyto) ribosomal precursor particles. Ribosome biogenesis intermediates containing the chromosomally TAP-tagged version of the indicated biogenesis factors were isolated by TAP purifications from cells transformed with plasmids expressing HA-Jjj1 or HA–Jjj1-ΔC. After isolation and elution from the beads, proteins were analyzed by SDS-PAGE and immunoblotting (IB). (B) Association of Zuo1 with nuclear (Nu) and cytoplasmic (Cyt) ribosomal precursor particles. Zuo1-containing complexes were isolated from cells containing the chromosomally TAP-tagged version of the indicated biogenesis factors and transformed with a plasmid expressing Zuo1-GFP. The presence of ribosome biogenesis intermediates in the GFP immunoprecipitation (IP) was assessed and analyzed by SDS-PAGE, TAP, and GFP immunoblotting.

Figure 7.

Jjj1 and Zuo1 play early and distinct roles in ribosome biogenesis. (A) Schematic representation of biogenesis of eukaryotic ribosomal rRNA maturation particles. The position of endonucleolytic cleavage steps and the subcellular localization of each step are indicated. (B) Loss of Jjj1 blocks a nuclear step of rRNA processing. rRNA processing microarray data (obtained from Peng et al., 2003) extracted to show the genes clustering together with Jjj1. The microarray data from Rei1 and Arx1 deletions are also included for comparison. The regions enriched in the jjj1 deletion strain, reflecting slower processing respect to WT, are highlighted in yellow. (C) rRNA processing defects in Δjjj1, Δzuo1, and Δssb1/2 cells measured by microarray experiments as described previously (Peng et al., 2003) using the indicated rRNA probes. Log2 fold induction of the indicated mutants over WT was calculated from triplicate experiments. (D) Loss of Zuo1–Ssz1 (RAC) blocks a nuclear step of rRNA processing. Pulse-chase labeling with [³H]uracil was performed with the WT and ΔRAC cells. Cells were pulse labeled for 2 min and chased as indicated with an excess of cold uracil. Total labeled RNAs were purified, separated on a denaturing agarose gel, and autoradiographed. The positions of the intermediate and mature rRNAs are indicated.

Figure 8.

Schematic representation of the role of Jjj1 and the Zuo1–Ssz1–SSB network in ribosome biogenesis. Major ribosomal maturation intermediates and rRNA processing steps are schematically indicated. Jjj1 and Zuo1 bind to nuclear assembly intermediates and regulate the activity of SSA and SSB, respectively. Jjj1 and Zuo1 bind first to nuclear 60S intermediates in the nucleus and stay bound to the mature cytoplasmic 60S ribosomal subunit. Red, Jjj1; orange, SSA; purple, Zuo1; blue, SSB. See Discussion for details.