A Noc Complex Specifically Involved in the Formation and Nuclear Export of Ribosomal 40 S Subunits

Abstract

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A 0 , A 1 , and A 2 . Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.Eukaryotic ribosome biogenesis is spatially organized into different subcellular compartments. Most steps in the pathway leading to mature ribosomes occur in the nucleolus, a specialized nuclear substructure, which includes transcription of the rDNA by RNA polymerase I, modification of the synthesized precursor RNA, and the assembly of both many ribosomal and non-ribosomal proteins with pre-ribosomal RNA (1). In the yeast Saccharomyces cerevisiae the resulting large ribonucleoprotein complex forms the 90 S pre-ribosome, which is split into precursor particles for the mature 40 S and 60 S ribosomal subunit (2). During or after their maturation the pre-ribosomes leave the nucleolus, move toward the nuclear pore, gain export competence, and are finally exported into the cytoplasm. Some maturation steps like processing of the 20 S rRNA intermediate within the 40 S subunit and the association of a few ribosomal proteins to the ribosomes occur rather late, even in the cytoplasm (3).Many factors known to be involved in biosynthesis and maturation of ribosomes were identified and characterized in S. cerevisiae (4,5). This organism represents a well suited model organism to study eukaryotic ribosome biogenesis, because homologues of the factors required are found in many eukaryotes. Of the more than 70 non-ribosomal proteins that participate in generation of ribosomes, most have been described to be required for modification of rRNA or removal of the external and internal spacer sequences from the precursor 35 S pre-RNA. End products of the rRNA processing pathways are the 18 S rRNA, which is present in the 40 S subunit and the 25 S and 5.8 S rRNA, as well as the RNA polymerase III-encoded 5 S rRNA, which are the rRNA constituents of the 60 S subunit. Among the transacting factors involved to produce mature 40 S and 60 S subunits are nucleases, putative RNA helicases, RNA modifying proteins, and proteins associated with small nucleolar RNAs (4, 5) (see also www.expasy.ch/linder/proteins.html).Folding, processing, and maturation of the rRNA is coordinated with the association of ribosomal proteins, with the assembly and disassembly of tran...

A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits

“…All of these proteins are essential for viability and conserved from yeast to humans, indicating that they play important functions in ribosome synthesis. Noc proteins have already been implicated in ribosomal export (Milkereit et al 2001(Milkereit et al , 2003, and we have successfully built 3D models that confirm their similarity to HEAT-repeat proteins (M. Dlakić and D. Tollervey, in prep. ).…”

“…We report here that Rrp12p, Upt10p, and Utp20p are each predicted to consist of HEAT-repeat motifs, as are five other ribosome synthesis factors including Noc2p, Noc3p, and Noc4p, which are known to be required for subunit export (Milkereit et al 2001(Milkereit et al , 2003. We propose that these large, extended proteins play key roles in ribosome synthesis by providing a framework for pre-ribosome assembly and participate in subunit export to the cytoplasm.…”

Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

“…For RNA analysis, cells were grown similarly except that the raffinose medium contained no glucose and that cells were washed twice with prewarmed galactose medium without CuSO 4 . The following described plasmids were used in this study: pFA6a-TRP1-PGAL1-GFP, pFA6a-TRP1-PGAL1-3HA (14), pRS316-RPL25-GFP (18), pRS314-RPS2-GFP (19). Plasmid pYCG-YLR106c was obtained from EUROSCARF.…”

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…In addition, ribosomal gene transcription is regulated through the modulation of the transcriptional apparatus and epigenetic silencing (131). Large and small mature ribosome particles are independently exported to the cytoplasm through an exportin 1 (CRM1) and Ran-GTP-dependent mechanism: export of 60S subunit requires the exchange of complexes Noc1-Noc2 by Noc3-Noc2 (102) and the association with the adaptor shuttling protein NMD3 (142), whereas the 40S needs the heterodimer Noc4p/Nop14p (103). The work by Hinsby et al exploited a machine learning-based predictor of nuclear export signals to analyze the late stage pre-40S complex, suggesting a role also for the human homolog of yeast DIM2p in the targeting and translocation of the late 40S to the cytoplasm (63).…”