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Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre-ribosomal particles. We report rRNA-binding sites for six late-acting 40S ribosome synthesis factors, three of which cluster around the 30 end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in 'beak' structure formation during 40S maturation-and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1- and Tsr1-binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3'-endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre-rRNA. Nob1 binds before pre-rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.

Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli , GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.

Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.

A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 335 exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6 were confirmed for rrp47 and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of “cryptic” noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism. The results of genetic and biochemical analyses indicate that the exosome 3-to-5 exonuclease complex has potent in vivo RNA degradation activity, even on highly structured RNAs and large RNA-protein (RNP) complexes. However, the purified eukaryotic exosome shows limited activity in vitro, and this

During transcription termination by RNA polymerase II on protein-coding genes, the nuclear 5′ exonuclease Rat1/Xrn2 degrades the nascent transcript downstream from the polyadenylation site and “torpedoes” the polymerase. We report that the activity of Rat1 is also required for efficient termination by RNA polymerase I (Pol I) on the rDNA. In strains lacking catalytically active Rat1 or its cofactor Rai1, Pol I reads through the major, “Reb1-dependent” terminator (T1) but stops downstream at the “fail-safe” terminator (T2) and replication fork barrier (RFB). The absence of both Rat1 and the RFB-binding protein Fob1 increased Pol I read-through of T2 and the RFB. We propose that cotranscriptional cleavage of the pre-rRNA by the endonuclease Rnt1 generates a loading site for the Rat1/Rai1 complex, which then degrades the nascent transcript. When Rat1 catches Pol I, which is predicted to be paused at T1, transcription is terminated.

Small nucleolar RNAs (snoRNAs) associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, which are essential for ribosome biogenesis. The snoRNAs are transcribed, processed, and assembled in snoRNPs in the nucleoplasm. Mature particles are then transported to the nucleolus. In yeast, 3′-end maturation of snoRNAs involves the activity of Rnt1p endonuclease and cleavage factor IA (CFIA). We report that after inhibition of CFIA components Rna14p and Rna15p, the snoRNP proteins Nop1p, Nop58p, and Gar1p delocalize from the nucleolus and accumulate in discrete nucleoplasmic foci. The U14 snoRNA, but not U3 snoRNA, similarly redistributes from the nucleolus to the nucleoplasmic foci. Simultaneous depletion of either Rna14p or Rna15p and the nuclear exosome component Rrp6p induces accumulation of poly(A)+ RNA at the snoRNP-containing foci. We propose that the foci detected after CFIA inactivation correspond to quality control centers in the nucleoplasm.

Summary The creAd-30 mutation leading to carbon catabolite derepression in Aspergillus nidulans is a pericentric inversion, having one breakpoint within the creA gene on the left arm of chromosome I and the other breakpoint between binG and yA on the right arm. The left-arm breakpoint alters the creA transcript. The likelihood that the inversion truncates creA centrally strengthens a previous proposal that derepression is the phenotype of loss-of-function mutations in creA.

Proteomic analyses in yeast have identified a large number of proteins that are associated with preribosomal particles. However, the product of the yeast ORF YJL010C, herein designated as Nop9, failed to be identified in any previous physical or genetic analysis of preribosomes. Here we report that Nop9 is a nucleolar protein, which is associated with 90S and 40S preribosomes. In cells depleted of Nop9p, early cleavages of the 35S pre-rRNA are inhibited, resulting in the nucleolar retention of accumulated precursors and a failure to synthesize 18S rRNA. Nop9 contains multiple pumilio-like putative RNA binding repeats and displays robust in vitro RNA binding activity. The identification of Nop9p as a novel, essential factor in the nuclear maturation of 90S and pre-40S ribosomal subunits shows that the complement of ribosome synthesis factors remains incomplete.

The TRAMP polyadenylation complexes have well-established functions in RNA surveillance, stimulating degradation by the 3′ to 5′ exonuclease activity of the exosome on a wide range of nuclear RNAs and RNA–protein complexes. Known targets include some of the non-protein coding RNA transcripts (ncRNAs), which are apparently widely transcribed from yeast and mammalian genomes. We will discuss potential mechanisms of TRAMP recruitment and exosome activation during RNA surveillance and degradation. Less well-understood observations link both the TRAMP and exosome complexes to chromatin structure and DNA repair, and we will speculate on the potential significance of these activities.

A family of HEAT-repeat containing ribosome synthesis factors was previously identified in Saccharomyces cerevisiae. We report the detailed characterization of two of these factors, Utp10 and Utp20, which were initially identified as components of the small subunit processome. Coprecipitation analyses confirmed the association of Utp10 and Utp20 with U3 snoRNA and the early pre-rRNA processing intermediates. Particularly strong association was seen with aberrant processing intermediates, which may help target these RNAs for degradation. Genetic depletion of either protein inhibited the early pre-rRNA processing steps in 18S rRNA maturation but had little effect on pre-rRNA transcription or synthesis of the 25S or 5.8S rRNAs. The absence of the poly(A) polymerase Trf5, a component of the TRAMP5 complex and exosome cofactor, led to stabilization of the aberrant 23S RNA in strains depleted of Utp10 or Utp20. In the case of Utp10, 20S pre-rRNA synthesis was also modestly increased by this loss of surveillance activity.

In this paper we present a quantified model concerning the synthesis of pre-rRNAs. The chemical kinetics simulation software Dizzy has been chosen as both the modelling and simulation framework of our study. We discuss the validation of the model against the available experimental data and we show some preliminary results obtained from the study of our model. All the analyses are based on stochastic simulation.

Copyright © 2007, American Society for Microbiology. All Rights Reserved, Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome., T.C. was the recipient of a fellowship from Fundação para a Ciência e Tecnologia, Portugal. This work was supported by the European Commission QLG2-CT-2001-01554. D.T. and L.M. were supported by the Wellcome Trust.

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins., U3 snoRNA binds to pre-rRNA, helping to orchestrate key steps in ribosome assembly. This study identifies Dhr1 as the essential RNA helicase that releases U3 snoRNA and allows ribosome maturation to continue., Author Summary Ribosomes are intricate assemblies of RNA and protein that are responsible for decoding a cell’s genetic information. Their assembly is a very rapid and dynamic process, requiring many ancillary factors in eukaryotic cells. One critical factor is the U3 snoRNA, which binds to the immature ribosomal RNA to direct early processing and folding of the RNA of the small subunit. Although U3 is essential to promote assembly, it must be actively removed to allow completion of RNA folding. Such RNA dynamics are often driven by RNA helicases, and here we use a broad range of experimental approaches to identify the RNA helicase Dhr1 as the enzyme responsible for removing U3 in yeast. A combination of techniques allows us to assess what goes wrong when Dhr1 is mutated, which parts of the RNA molecules the enzyme binds to, and how Dhr1 unwinds its substrates.

Recent years have seen a dramatic increase in the number of ribosome synthesis factors identified in the yeast Saccharomyces cerevisiae. Most of these are not predicted to directly catalyze either RNA processing or modification, and they are therefore predicted to function in some sense as assembly factors, promoting the assembly and/or disassembly of the processing and modification machinery, binding of the ribosomal proteins and correct folding of the pre-rRNAs and rRNAs. In contrast, ribosome synthesis in E. coli, which has also been extensively analyzed, appears to involve a very small number of potential assembly factors. Here we will consider the differences between eukaryotic and bacterial ribosome synthesis that may underlie this distinction.

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.

The exosome complex of 3'-->5' exonucleases is an important component of the RNA-processing machinery in eukaryotes. This complex functions in the accurate processing of nuclear RNA precursors and in the degradation of RNAs in both the nucleus and the cytoplasm. However, it has been unclear how different classes of substrate are distinguished from one another. Recent studies now provide insights into the regulation and structure of the exosome, and they reveal striking similarities between the process of RNA degradation in bacteria and eukaryotes.

Microarray analyses were performed on yeast strains mutant for the nuclear-specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37 degrees C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA-binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5' fragments of the mRNA. These contain a predicted Nrd1p-binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read-through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read-through transcripts into downstream ORFs are normally rapidly degraded by the exosome.

Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.

A newly identified step in maturation of the small ribosomal particle regulated by the kinase Hrr25 is critical, for without Hrr25, immature 40S subunits accumulate. The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments1,2,3,4. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed5,6,7,8,9,10,11,12. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the ‘beak’. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Recent analyses have shown that the activity of the yeast nuclear exosome is stimulated by the Trf4p–Air1/2p–Mtr4p polyadenylation (TRAMP) complex. Here, we report that strains lacking the Rrp6p component of the nuclear exosome accumulate polyadenylated forms of many different ribosomal RNA precursors (pre-rRNAs). This polyadenylation is reduced in strains lacking either the poly(A) polymerase Trf4p or its close homologue Trf5p. In contrast, polyadenylation is enhanced by overexpression of Trf5p. Polyadenylation is also markedly increased in strains lacking the RNA helicase Mtr4p, indicating that it is required to couple poly(A) polymerase activity to degradation. Tandem affinity purification-tagged purified Trf5p showed polyadenylation activity in vitro, which was abolished by a double point mutation in the predicted catalytic site. Trf5p co-purified with Mtr4p and Air1p, indicating that it forms a complex, designated TRAMP5, that has functions that partially overlap with the TRAMP complex.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)(+) mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6 Delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome. mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

In addition to the genes that encode messenger ribonucleic acids and are translated into proteins, the human genome includes hundreds to thousands of genes that encode a variety of functional ribonucleic acid (RNA) molecules. The number of known RNA species has grown rapidly in recent years and many additional examples will doubtless be found. Keywords: tRNA; rRNA; snRNA; snoRNA; ribozyme

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Ase1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation. (c) 2005 Elsevier Inc. All rights reserved.

Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.

With the availability of the complete yeast genomic sequence, techniques which allow the rapid functional analysis of genes of interest are of increasing importance. Here we report a technique which allows the initial characterisation of genes of interest, through the construction of conditionally expressed mutations for functional analyses and the generation of epitope-tagged fusion proteins for immuno-localisation and immuno-purification, entirely by PCR.

Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified. Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar. Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3. Here, an in vitro processing system that accurately reproduces this cleavage is described. Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP. This establishes a role for RNase MRP in the nucleolus.

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.

Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)(+) RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5' degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in Ism mutants remain 5' capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.

Dcs1, the m(7)GpppX pyrophosphatase of Saccharomyces cerevisiae, has been reported to 'scavenge' capped 51 end fragments generated by 3'-->5' mRNA degradation. We now show that the absence of Dcs1, and the closely related Dcs2 protein, compromises cellular responses to glucose-deprivation stress as well as to step changes in glucose availability. Dcs1 and Dcs2 form homo- and heterodimers, with the heterodimer appearing as cells enter diauxie. Despite the previously observed increase in abundance of the mRNA encoding the neutral trehalase (Nth1) in the stationary phase, the total enzyme activity of Nth1 decreases in this phase of growth. Changes in trehalase activity are significant because the non-reducing disaccharide trehalose is thought to stabilize cellular components under stress conditions. In the dcs1Delta and dcs1Deltadcs2Delta mutants, normal regulation of trehalase activity is lost. Nutrient stress induces DCS1 and DCS2 transcription via the cAMP-PKA signalling pathway. Dcs1 also becomes phosphorylated as the availability of glucose diminishes, and we test the role of this phosphorylation in the stress response. Further evidence indicates that Dcs1 plays a complementary role to the translation factor eIF4E in preventing capped 5' fragments of mRNA from interfering with translation initiation. We conclude that Dcs1 function influences cellular responses to changes in nutrient availability, while Dcs2 seems to act as a modulator of Dcs1 function.

Nop15p is an essential protein that contains an RNA recognition motif (RRM) and localizes to the nucleoplasm and nucleolus. Cells depleted of Nop15p failed to synthesize the 25S and 5.8S rRNA components of the 60S ribosomal subunit, and exonucleolytic 5′ processing of 5.8S rRNA was strongly inhibited. Prer‐RNAs co‐precipitated with tagged Nop15p confirmed its association with early pre‐60S particles and Nop15p bound a pre‐rRNA transcript in vitro. Nop15p‐depleted cells show an unusually abrupt growth arrest prior to substantial depletion of ribosomal subunits. Following cell synchronization in mitosis, Nop15p‐depleted cells undergo nuclear division with wild‐type kinetics, activate the mitotic exit network and disassemble their mitotic spindle. However, they uniformly arrest at cytokinesis and fail to assemble a contractile actin ring at the bud neck. In dividing wild‐type cells, segregation of nucleolar proteins to the daughter nuclei occurs after separation of the nucleoplasm. In these late mitotic cells, Nop15p was partially delocalized from the nucleolus to the nucleoplasm, consistent with a specific function in cell division in addition to its role in ribosome synthesis.

Cic1p/Nsa3p was previously reported to be associated with the 26S proteasome and required for the degradation of specific substrates, but was also shown to be associated with early pre-60S particles and to be localized to the nucleolus. Here we report that Cic1p/Nsa3p is required for the synthesis of 60S ribosome subunits. A temperature-sensitive lethal cic1–2 point mutation inhibits synthesis of the mature 5.8S and 25S rRNAs. Release of the pre-60S particles from the nucleolus to the nucleoplasm was also inhibited as judged by the nuclear accumulation of an Rpl11b-GFP reporter construct. We suggest that Cic1p/Nsa3p associates early with nascent preribosomal particles and is required for correct processing and nuclear release of large ribosomal subunit precursors.

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.

The yeast ubiquitin-protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis. Here, we identify Rsp5p in a screen for tRNA export (tex) mutants. The tex23-1/rsp5-3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre-ribosomal subunits. In contrast, nuclear localization sequence (NLS)-mediated nuclear protein import is unaffected in this mutant. Strikingly, the nuclear RNA export defects seen in the rsp5-3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast. These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways.

Related exosome complexes of 335 exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg 2 -labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 335 exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3 processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47- strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3-extended nuclear pre-mRNAs or the cytoplasmic 335 mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs. The eukaryotic 18S, 5.8S, and 25S rRNAs (yeast nomenclature is given) are generated from a single large RNA polymerase I transcript by a series of endonucleolytic and exonucleolytic RNA processing reactions (reviewed in reference 39). The earliest detectable transcript in yeast, the 35S precursor rRNA (pre-rRNA), also undergoes extensive posttranscriptional modification involving predominantly pseudouridine formation and ribosyl-2-O-methylation. These modifications are directed to specific nucleotides within the 7-kb-long 35S prerRNA via complementary base-pairing mechanisms involving 70 different small nucleolar RNAs (snoRNAs).

Many different RNA species undergo nucleotide modifications at sites identified by guide small nucleolar ribonucleoprotein (snoRNP) particles. The co-crystal structure of two snoRNP proteins gives valuable clues into the workings of this system.

Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3' processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing. The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p.

In this issue of Molecular Cell, single-cell analyses by Bumgarner et al. (2012) reveal how two antagonistic long noncoding RNAs at the FLO11 locus define a toggle responsible for morphological heterogeneity in genetically identical populations of budding yeast.

Box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) contain four essential proteins, Cbf5p, Gar1p, Nhp2p, and Nop10p, each of which, with the exception of Gar1p, is required for box H/ACA snoRNA accumulation. Database searches identified a novel essential protein, which we termed Naf1p, with a region of homology to the RNA-binding domain of Gar1p and other features in common with hnRNP-like proteins. Naf1p is localized to the nucleus and is not a stable component of the H/ACA snoRNPs, but it is required for the accumulation of all box H/ACA snoRNAs. This requirement is not at the level of snoRNA transcription initiation or termination. Naf1p shows in vitro RNA-binding activity and also binds directly to Cbf5p and Nhp2p. Naf1p was shown to bind to the CTD in vivo in a two-hybrid assay, and the phosphorylated CTD, but not the nonphosphorylated CTD, was shown to precipitate tagged Naf1p from a cell lysate. We propose that Naf1p is recruited to the CTD of RNA polymerase II and binds to nascent box H/ACA snoRNAs promoting snoRNP assembly.

60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we,have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

The past year has seen dramatic changes in our understanding of ribosome synthesis, fuelled largely by advances in proteomic analysis. It is now possible to outline the pathway of ribosome assembly, which is highly dynamic and involves a remarkable separation of the factors involved in the synthesis of the 40S and 60S ribosomal subunits. Around 140 identified, non-ribosomal proteins are currently implicated in post-transcriptional ribosome synthesis in yeast.

Depletion of any of the five essential proteins Lsm2p to Lsm5p and Lsm8p leads to strong accumulation of all tested unspliced pre-tRNA species, as well as accumulation of 5' and 3' unprocessed species. Aberrant 3'-extended pre-tRNAs were detected, presumably due to stabilization of transcripts that fail to undergo correct transcription termination, and the accumulation of truncated tRNA fragments was also observed. Tandem affinity purification-tagged Lsm3p was associated with pre-tRNA primary transcripts and, less efficiently, with other unspliced pre-tRNA intermediates but not mature tRNAs. Association of the Saccharomyces cerevisiae La homologue Lhp1p with pre-tRNAs was reduced approximately threefold on depletion of Lsm3p or Lsm5p. The association of Lhp1p with larger RNA polymerase III transcripts, pre-RNase P RNA and the signal recognition particle RNA (scR1), was more drastically reduced. The impaired pre-tRNA processing seen on Lsm depletion is not, however, due solely to reduced Lhp1p association as evidenced by analysis of lhp1-Delta strains depleted of Lsm3p or Lsm5p. These data are consistent with roles for an Lsm complex as a chaperone that facilitates the efficient association of pre-tRNA processing factors with their substrates.

The structure of a ribosome assembly factor in complex bound to a ribosomal protein uncovers a chaperoning function that uses RNA mimicry and is regulated by ATP hydrolysis., During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation., Author Summary Ribosomes are the cellular machines responsible for all protein synthesis. In eukaryotes, the assembly of ribosomes from their protein and RNA components is extremely complicated and involves more than 200 nonribosomal factors—three times the number of proteins in the mature complex. Among these factors, the Fap7 protein is particularly intriguing because it interacts with the small subunit ribosomal protein Rps14 and it exhibits adenylate kinase activity—a molecular function more commonly associated with regulating ATP/ADP levels than assembling protein–RNA complexes. Combining structural and biochemical analysis of the Rps14–Fap7 complex, we show that Fap7 uses protein side chains to mimic RNA contacts, rendering the interaction of Rps14 with ribosomal RNA or with Fap7 competitive and mutually exclusive. Once bound, Rps14 blocks the substrate-binding cavity of Fap7, and ATP hydrolysis will then break the Fap7–Rps14 complex apart. At the same time, the ribosome structure at the location where Rps14 binds is disrupted when the Fap7/Rps14 complex is formed, and this process is regulated by ATP binding and hydrolysis. Our model is thus that Fap7 temporarily removes Rps14 from the ribosome to enable a conformational change of the ribosomal RNA that is needed for the final maturation step of the small ribosomal subunit.

During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC). These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5′-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of "Miller" chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼1.5Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.

International audience; Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rp125p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits, We find that thermosensitive nmd3 mutants are impaired ill large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.