RESUMEN
To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the 'B-factor' proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes.
Asunto(s)
Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ARN Helicasas DEAD-box/metabolismo , GTP Fosfohidrolasas , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Eliminación de Gen , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Proteínas Nucleares/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.