Ytm1, Nop7, and Erb1 Form a Complex Necessary for Maturation of Yeast 66S Preribosomes

Abstract

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 27SA 2 , 27SA 3 , 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.Biogenesis of eukaryotic ribosomes is a highly regulated and dynamic process that begins in the nucleolus with transcription of a precursor rRNA (pre-rRNA) that is rapidly packaged into the 90S ribonucleoprotein particle containing ribosomal proteins, nonribosomal proteins, and snoRNA-containing ribonucleoprotein particles (snoRNPs). The 90S pre-RNPs are converted into 43S and 66S ribosome assembly intermediates, which ultimately give rise to mature 40S and 60S ribosomal subunits (Fig. 1).Molecular genetic approaches in yeast identified more than 70 trans-acting factors required for ribosome assembly (12,14,46). Subsequent advances in proteomics enabled purification of pre-rRNPs from yeast and identification of an additional 80 assembly factors present in preribosomes, as well as most of those proteins previously discovered using genetic screens (3,7,11,17,20,21,24,26,37,38,41,(49)(50)(51). Metazoan homologues of most of the yeast ribosome assembly factors were discovered by proteomic analysis of purified nucleoli (2, 52).Among the assembly factors found in yeast preribosomes are 17 proteins containing WD40 motifs (14). These motifs function as protein-protein interaction domains (53). Therefore, such WD40-containing proteins may nucleate assembly of preribosomes by interacting sequentially or simultaneously with other assembly factors or ribosomal proteins. Previously, we identified the WD40 protein Ytm1 as a constituent of purified 66S pre-rRNPs and showed that depletion of Ytm1 results in a deficiency of 60S ribosomal subunits (21).In this study, we have further investigated the role of Ytm1 in ribosome biogenesis. Ytm1 is a constituent of multiple consecutive 66S preribosomes containing 27SA 2 , 27SA 3 , 27SB, 25.5S, and 7S pre-rRNAs plus a collection of ribosomal and nonribosomal proteins. Ytm1 is present in a heterotrimer with two other assembly factors, Nop7 and Erb1, both within 66S pre-rRNPs and as a subcomplex independent of preribosomes. Mutations in Ytm1 disrupt interactions between Ytm1 and Erb1, destabilize the heterotrimer, and significantly reduce association of these three proteins with 66S preribosomes. These 66S pre-rRNPs ...

Interactions among Ytm1, Erb1, and Nop7 Required for Assembly of the Nop7-Subcomplex in Yeast Preribosomes

“…Two-hybrid assays and GST pulldown experiments show that Nop7 and Ytm1 interact directly with Erb1, but not with each other (Miles et al, 2005). Mammalian counterparts of these three proteins, Pes1, WDR12, and Bop1, also associate with each other to form the PeBoW complex (Hö lzel et al, 2005).…”

“…Knock-down of the mammalian proteins also affects processing of the corresponding pre-rRNAs. Nop7 and Erb1 assemble into 90S preribosomal particles containing 35S pre-rRNA, whereas Ytm1 is recruited later, into 66S A 2 preribosomes (Miles et al, 2005). Thus, Nop7, Ytm1, and Erb1 function several steps after they associate with preribosomes, suggesting that there may be separate means to recruit and to utilize these proteins in subunit assembly.…”

“…Identification of networks of interactions among the assembly factors, ribosomal proteins, and ribosomal RNAs is an important first step to address this question. To begin to identify this internal structure of preribosomes and to connect this structural information to function of ribosomal molecules, we have used a combined genetic and biochemical approach to identify neighborhoods or subcomplexes of interacting proteins within preribosomes (Harnpicharnchai et al, 2001;Miles et al, 2005;Zhang et al, 2007).…”

“…One such neighborhood is the Nop7-subcomplex, which contains assembly factors Nop7, Ytm1, and Erb1 (Harnpicharnchai et al, 2001;Du and Stillman, 2002;Krogan et al, 2004, Miles et al, 2005. Two-hybrid assays and GST pulldown experiments show that Nop7 and Ytm1 interact directly with Erb1, but not with each other (Miles et al, 2005).…”

“…Each of these proteins is essential, conserved among eukaryotes, located primarily in the nucleolus, and required for the assembly of 60S ribosomal subunits (Strezoska et al, 2000;Pestov et al, 2001;Adams et al, 2002;Lerch-Gaggl et al, 2002;Oeffinger et al, 2002;Miles et al, 2005;Hö lzel et al, 2005). The Nop7-subcomplex is also a member of a "functional cluster" in yeast; depletion of each of these three proteins causes identical defects in rRNA precursor (pre-rRNA) processing: altered conversion of 27SA 3 pre-rRNA to 27SB S pre-rRNA (Adams et al, 2002;Miles et al, 2005;Oeffinger et al, 2002;Pestov et al, 2001). Knock-down of the mammalian proteins also affects processing of the corresponding pre-rRNAs.…”