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.

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.

Previous work from our lab suggests that a group of interdependent assembly factors (A 3 factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A 3 factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA 3 pre-rRNA processing, even though the A 3 factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A 3 factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures. Ribosome assembly is a highly conserved and dynamic process driven by the cooperative transcription, folding, modification, and processing of rRNAs and stable binding of ribosomal proteins (r-proteins) (1, 2). In Saccharomyces cerevisiae, this process begins in the nucleolus with cotranscriptional assembly of early precursor particles. As transcription proceeds, the nascent pre-rRNA is cleaved, separating maturation of early 40S and 60S precursors. A series of endo-and exonucleolytic cleavages remove internal and external transcribed spacer sequences from prerRNAs, as the assembling ribosome moves from the nucleolus to the nucleoplasm and is eventually exported to the cytoplasm ( Fig. 1A and B).Assembly of yeast ribosomes requires ϳ180 trans-acting proteins termed assembly factors (AFs). The majority of these proteins are conserved across eukaryotes, are essential for cell growth, and are thought to function as scaffolding proteins, RNA chaperones, energy-consuming nucleoside triphosphatases (NTPases), nucleases, or posttranslational modifiers (3-5). Purification of ribosome assembly intermediates allowed the identification of most AFs, and initial characterizations have shown in which steps of pre-rRNA processing many of these factors function. However, to begin to understand the function of these proteins on a mechanistic level, one must address the following: the timing of association of these proteins with preribosomes; how they are recruited to preribosomes; their requirement for the stable binding of other proteins; and their role in folding rRNA.Numerous studies have shown that subsets of AFs can be isolated as subcomplexes (6-23). Proteins within a subcomplex are often required for the same steps of pre-rRNA processing, suggesting that they function together during ribosome bi...

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