Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis Giulia Moriggi1, Blanca Nieto1, Mercedes Dosil1,2* 1 Centro de Investigacio´n del Ca´ncer and Instituto de Biologı´a Molecular y Celular del Ca´ncer (IBMCC), CSIC-University of Salamanca, Salamanca, Spain, 2 Departamento de Bioquı´mica y Biologı´a Molecular, University of Salamanca, Salamanca, Spain
Abstract During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes. Citation: Moriggi G, Nieto B, Dosil M (2014) Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis. PLoS Genet 10(12): e1004836. doi:10.1371/journal.pgen.1004836 Editor: Herbert Tschochner, University of Regensburg Germany, Germany Received May 6, 2014; Accepted October 17, 2014; Published December 4, 2014 Copyright: ß 2014 Moriggi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work is supported by grants from both the Spanish Ministry of Economy and Competitiveness (BFU2011-23668, RD06/0020/0001 and RD12/0036/ 0002), the Samuel Solo´rzano Barruso Foundation (FS/17-2013) and the Castilla y Leo´n Autonomous Government (CSI039A12-1). GM and BN have been supported by graduate student contracts by the University of Salamanca and Santander Bank and, in the case of BN, by the RD06/0020/0001 grant. Spanish funding is cosponsored by the European Union FEDER Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]
of the non-ribosomal components originally present in the 90S pre-ribosome and the rapid degradation of processing byproducts [2]. The early pre-60S particles contain .40 associated factors, and undergo multiple maturation steps that are accompanied by major changes in composition until exiting the nucleus [2,10,11,12]. In contrast, the early pre-40S particles are thought to have a relatively low compositional complexity and are rapidly exported, consistent with the fact that the 20S pre-rRNA is not further processed inside the nucleus (Figure 1A) [2,10,11,13]. Final maturation of 40S subunits, which includes a proofreading step through association to 60S subunits and the cleavage of the 20S pre-rRNA at site D, takes place in the cytoplasm [14,15,16,17]. Due to the rapid kinetics of transit thorough the nucleoplasm, it is assumed that the major events of pre-40S particle assembly take place concurrently with the 35S pre-RNA cleavage steps in the nucleolus. Despite this, the pre-40S particles released from 90S preribosomes have to undergo some transformations before leaving the nucleus. These include the recruitment of factors that will participate in cytoplasmic maturation processes as well as transport proteins involved in particle transit through nuclear pores [18,19]. Pre-40S particles are also known to
Introduction The formation of ribosomes in eukaryotic cells requires the production and subsequent assembly of four rRNAs and
Abstract During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes. Citation: Moriggi G, Nieto B, Dosil M (2014) Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis. PLoS Genet 10(12): e1004836. doi:10.1371/journal.pgen.1004836 Editor: Herbert Tschochner, University of Regensburg Germany, Germany Received May 6, 2014; Accepted October 17, 2014; Published December 4, 2014 Copyright: ß 2014 Moriggi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work is supported by grants from both the Spanish Ministry of Economy and Competitiveness (BFU2011-23668, RD06/0020/0001 and RD12/0036/ 0002), the Samuel Solo´rzano Barruso Foundation (FS/17-2013) and the Castilla y Leo´n Autonomous Government (CSI039A12-1). GM and BN have been supported by graduate student contracts by the University of Salamanca and Santander Bank and, in the case of BN, by the RD06/0020/0001 grant. Spanish funding is cosponsored by the European Union FEDER Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]
of the non-ribosomal components originally present in the 90S pre-ribosome and the rapid degradation of processing byproducts [2]. The early pre-60S particles contain .40 associated factors, and undergo multiple maturation steps that are accompanied by major changes in composition until exiting the nucleus [2,10,11,12]. In contrast, the early pre-40S particles are thought to have a relatively low compositional complexity and are rapidly exported, consistent with the fact that the 20S pre-rRNA is not further processed inside the nucleus (Figure 1A) [2,10,11,13]. Final maturation of 40S subunits, which includes a proofreading step through association to 60S subunits and the cleavage of the 20S pre-rRNA at site D, takes place in the cytoplasm [14,15,16,17]. Due to the rapid kinetics of transit thorough the nucleoplasm, it is assumed that the major events of pre-40S particle assembly take place concurrently with the 35S pre-RNA cleavage steps in the nucleolus. Despite this, the pre-40S particles released from 90S preribosomes have to undergo some transformations before leaving the nucleus. These include the recruitment of factors that will participate in cytoplasmic maturation processes as well as transport proteins involved in particle transit through nuclear pores [18,19]. Pre-40S particles are also known to
Introduction The formation of ribosomes in eukaryotic cells requires the production and subsequent assembly of four rRNAs and
