Many RNA nucleases and helicases participate in ribosome biogenesis, but how they cooperate with each other is largely unknown. Here we report that in vivo cleavage of the yeast pre-rRNA at site D, the 3-end of the 18 S rRNA, requires functional interactions between PIN (PilT N terminus) domain protein Nob1 and the DEAH box RNA helicase Prp43. Nob1 showed specific cleavage on a D-site substrate analogue in vitro, which was abolished by mutations in the Nob1 PIN domain or the RNA substrate. Genetic analyses linked Nob1 to the late pre-40 S-associated factor Ltv1, the RNA helicase Prp43, and its cofactor Pfa1. In strains lacking Ltv1, mutation of Prp43 or Pfa1 led to a striking accumulation of 20 S pre-rRNA in the cytoplasm due to inhibition of site D cleavage. This phenotype was suppressed by increased dosage of wildtype Nob1 but not by Nob1 variants mutated in the catalytic site. In ltv1/pfa1 mutants the 20 S pre-rRNA was susceptible to 3 to 5 degradation by the cytoplasmic exosome. This degraded into the 3 region of the 18 S rRNA, strongly indicating that the preribosomes are structurally defective.Eukaryotic ribosome formation is a complex process that requires coordination of rRNA synthesis and processing with the subsequent incorporation of the ribosomal (Rpl and Rps) proteins (reviewed in Refs. 1-5). In the yeast Saccharomyces cerevisiae, ribosome synthesis starts with the transcription of a 35 S pre-rRNA, which is the common precursor to the mature 18, 5.8, and 25 S rRNAs. This pre-rRNA is co-transcriptionally bound by ribosomal proteins as well as many non-ribosomal trans-acting factors to form a 90 S preribosomal particle. Next, this 90 S intermediate undergoes a series of early pre-rRNA processing and modification steps in the nucleolus before an endonucleolytic cleavage at site A 2 in the pre-rRNA separates the pathways for 60 and 40 S subunit assembly.Pre-60 S particles undergo further maturation steps in the nucleus, whereas pre-40 S particles contain few non-ribosomal proteins and are rapidly exported to the cytoplasm. The factors involved in 40 S export are largely unknown, but the non-essential pre-40 S factor Ltv1 was suggested to play a role in this process (6). Following nuclear export, the pre-40 S subunit undergoes cytoplasmic maturation, involving two major events. (i) 20 S pre-rRNA cleavage at site D generates the 3Ј-end of the mature 18 S rRNA, and (ii) structural reorganization forms the characteristic beak structure of the mature 40 S subunit, resulting in exposure of RNA helix 33 within the 18 S rRNA (7).The sites and the order of endonuclease and exonuclease processing events that convert the 35 S pre-rRNA into the mature rRNA species are well characterized in yeast (Fig. 3A) (reviewed in Refs. 4 and 8). However, although most or all of the exonucleases participating in rRNA processing are known, several predicted endonucleases remain to be identified. Two proteins, Fap7 and Nob1, were proposed to cleave site D (9 -11), but for neither was endonuclease activity shown. Fap7 is a putativ...

The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.

Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation.

Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter was complemented by UBA2 (a subunit of the E1 sumoylation enzyme). Mutations in UBC9 (E2 enzyme), ULP1 [small-ubiquitin-related modifier (SUMO) isopeptidase] and SMT3 (SUMO-1) caused 60S export defects. A directed analysis of the SUMO proteome revealed that many ribosome biogenesis factors are sumoylated. Importantly, preribosomal particles along both the 60S and the 40S synthesis pathways were decorated with SUMO, showing its direct involvement. Consistent with this, early 60S assembly factors were genetically linked to SUMO conjugation. Notably, the SUMO deconjugating enzyme Ulp1, which localizes to the nuclear pore complex (NPC), was functionally linked to the 60S export factor Mtr2. Together our data suggest that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.Key words: Mtr2, nuclear export, ribosome biogenesis, ribosome export, SUMO, Ulp1, yeast Smt3Received 12 May 2006, revised and accepted for publication 6 July 2006Eukaryotic ribosome biogenesis is a highly dynamic and coordinated multistep process that starts with transcription of rDNA (ribosomal DNA) repeats by RNA polymerase I [25S, 18S and 5.8S ribosomal RNA (rRNA)] and RNA polymerase III (5S rRNA). The RNA polymerase-I-derived transcript (35S pre-rRNA) undergoes extensive processing and modification (1,2). Concomitantly, ribosomal proteins are assembled onto the maturing rRNA to form preribosomal particles (3). In the past, genetic screens have identified many factors involved in the assembly of the ribosomal subunits (1). Recently, a major advance in the field was achieved by isolation of maturing preribosomal particles via tandem affinity purification (TAP) methods, aided by sensitive mass spectrometry. This greatly expanded the inventory of nonribosomal factors that participate in the assembly, maturation and transport of preribosomal particles (4-7). These factors include rRNAmodifying enzymes, endonucleases, exonucleases, RNA helicases, adenosine triphosphatases associated with various cellular activities, guanosine triphosphatases and proteins associated with small nucleolar RNAs (3). Subsequent proteomic approaches helped unravel changes in the protein and rRNA composition of preribosomal particles (60S and 40S) as they travel from their site of initial assembly in the nucleolus to their final site of maturation in the cytoplasm (6,8-10).We have previously described a visual assay to isolate factors involved in 60S export that detects the nucleolar/ nuclear accumulation of an Rpl25-eGFP reporter in thermosensitive (ts) mutants (11). By screening a ts mutant collection using this assay,...

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.