Interactions among Ytm1, Erb1, and Nop7 required for assembly of the Nop7-subcomplex in yeast preribosomes

In Saccharomyces cerevisiae, more than 180 assembly factors associate with preribosomes to enable folding of pre-rRNA, recruitment of ribosomal proteins, and processing of pre-rRNAs to produce mature ribosomes. To examine the molecular architecture of preribosomes and to connect this structure to functions of each assembly factor, assembly subcomplexes have been purified from preribosomal particles. The Nop7-subcomplex contains three assembly factors: Nop7, Erb1, and Ytm1, each of which is necessary for conversion of 27SA(3) pre-rRNA to 27SB(S) pre-rRNA. However, interactions among these three proteins and mechanisms of their recruitment and function in pre-rRNPs are poorly understood. Here we show that Ytm1, Erb1, and Nop7 assemble into preribosomes in an interdependent manner. We identified which domains within Ytm1, Erb1, and Nop7 are necessary for their interaction with each other and are sufficient for recruitment of each protein into preribosomes. Dominant negative effects on growth and ribosome biogenesis caused by overexpressing truncated Ytm1, Erb1, or Nop7 constructs, and recessive phenotypes of the truncated proteins revealed not only interaction domains but also other domains potentially important for each protein to function in ribosome biogenesis. Our data suggest a model for the architecture of the Nop7-subcomplex and provide potential functions of domains of each protein.     

Degradation of ribosomal RNA precursors by the exosome

The yeast exosome is a complex of 3′→5′ exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3′ end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A₀, A₁, A₂ and A₃, reducing the levels of the 32S, 20S, 27SA₂ and 27SA₃ pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3′→5′ exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans-acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A₂-C₂ RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.     

Ytm1, Nop7, and Erb1 form a complex necessary for maturation of yeast 66S preribosomes

The essential, conserved yeast nucleolar protein Ytm1 is one of 17 proteins in ribosome assembly intermediates that contain WD40 protein-protein interaction motifs. Such proteins may play key roles in organizing other molecules necessary for ribosome biogenesis. Ytm1 is present in four consecutive 66S preribosomes containing 27SA2, 27SA3, 27SB, and 25.5S plus 7S pre-rRNAs plus ribosome assembly factors and ribosomal proteins. Ytm1 binds directly to Erb1 and is present in a heterotrimeric subcomplex together with Erb1 and Nop7, both within preribosomes and independently of preribosomes. However, Nop7 and Erb1 assemble into preribosomes prior to Ytm1. Mutations in the WD40 motifs of Ytm1 disrupt binding to Erb1, destabilize the heterotrimer, and delay pre-rRNA processing and nuclear export of preribosomes. Nevertheless, 66S preribosomes lacking Ytm1 remain otherwise intact.     

Enp1, a yeast protein associated with U3 and U14 snoRNAs,is required for pre-rRNA processing and 40S subunit synthesis

ENP1 is an essential Saccharomyces cerevisiae gene encoding a 483 amino acid polypeptide. Enp1 protein is localized in the nucleus and concentrated in the nucleolus. An enp1-1 temperature-sensitive mutant inhibited 35S pre-rRNA early processing at sites A₀, A₁ and A₂ as shown by northern analysis of steady state levels of rRNA precursors. Pulse-chase analysis further revealed that the enp1-1 strain was defective in the synthesis of 20S pre-rRNA and hence 18S rRNA, which led to reduced formation of 40S ribosomal subunits. Co-precipitation analysis revealed that Enp1 was associated with Nop1 protein, as well as with U3 and U14 RNAs, two snoRNAs implicated in early pre-rRNA processing steps. These results suggest a direct role for Enp1 in the early steps of rRNA processing.     

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.     

Nop53p, an essential nucleolar protein that interacts with Nop17p and Nip7p, is required for pre-rRNA processing in Saccharomyces cerevisiae

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.     

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C₁ and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7^Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.     

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.     

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.     

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.     

All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S_B precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S_B precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A₀, A₁ and A₂. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.     

Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.     

Analysis of nucleolar pre-rRNA processing sites in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

The location of rRNA processing was analyzed by usingin situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular components (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed.     

Dim2p, a KH-domain protein required for small ribosomal subunit synthesis

Recent proteomic analyses are revealing the dynamics of preribosome assembly. Following cleavage at processing site A(2), which generates the 20S pre-rRNA (the immediate precursor to the 18S rRNA), early RRPs (ribosomal RNA processing factors) are released in bulk from the preribosomes, and the resulting pre-40S subunits are left associated with a limited set of proteins that we refer to as the SSU RRP complex. Dim2p, a core constituent of the SSU RRP complex and conserved KH-domain containing protein, is required for pre-rRNA processing and is associated with early nucleolar and late cytoplasmic pre-rRNA species. Consistently, Dim2p shuttles between the nucle(ol)us and the cytoplasm, a trafficking that is tightly regulated by growth. The association of Dim2p with the 18S rRNA dimethyltransferase Dim1p, as well as its requirement for pre-rRNA processing at cleavage sites A(1) and A(2) and for 18S rRNA dimethylation, suggest that Dim2p may recruit Dim1p to nucleolar pre-rRNAs through its KH domain.     

Nop9 is an RNA binding protein present in pre-40S ribosomes and required for 18S rRNA synthesis in yeast

Proteomic analyses in yeast have identified a large number of proteins that are associated with preribosomal particles. However, the product of the yeast ORF YJL010C, herein designated as Nop9, failed to be identified in any previous physical or genetic analysis of preribosomes. Here we report that Nop9 is a nucleolar protein, which is associated with 90S and 40S preribosomes. In cells depleted of Nop9p, early cleavages of the 35S pre-rRNA are inhibited, resulting in the nucleolar retention of accumulated precursors and a failure to synthesize 18S rRNA. Nop9 contains multiple pumilio-like putative RNA binding repeats and displays robust in vitro RNA binding activity. The identification of Nop9p as a novel, essential factor in the nuclear maturation of 90S and pre-40S ribosomal subunits shows that the complement of ribosome synthesis factors remains incomplete.