This study shows that Trm112 interacts with and is required for the presence of 18S rRNA methyltransferase Bud23. Also shown is the involvement of Trm112 in 60S biogenesis, thus extending the known functions of Trm112 from tRNA and translation factor methylation to roles in biogenesis of both ribosomal subunits.
We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.
Rps15p, an essential ribosomal protein, was previously shown to be critical for nuclear export of small subunit pre-particles. We have designed a synthetic lethal screen in Saccharomyces cerevisiæ to identify its genetic partners and further elucidate its role during ribosomal biogenesis. Our screen revealed interactions with mutants affected at various stages during ribosome biogenesis, from early nucleolar steps to nuclear export. Mutations were identified in genes encoding proteins involved in early ribosome biogenesis steps, like the small subunit processome component Utp15p, the 90S pre-ribosome factor Slx9p and the H/ACA snoRNP core protein Nhp2p. In addition, we found a synthetic lethality with BUD23, a gene encoding a methyltransferase involved both in rRNA modification and small subunit nuclear export. Interestingly, deletion of snR36 or snR85, two H/ACA snoRNAs that direct modifications close to Rps15p's binding site on the rRNA, produces mild and opposite effects on growth in an rps15 hypomorphic background. These data uncover an unreported link between a ribosomal protein and rRNA modification machinery.
Posttranscriptional and posttranslational modification of macromolecules is known to fine-tune their functions. Trm112 is unique, acting as an activator of both tRNA and protein methyltransferases. Here we report that in Saccharomyces cerevisiae, Trm112 is required for efficient ribosome synthesis and progression through mitosis. Trm112 copurifies with pre-rRNAs and with multiple ribosome synthesis trans-acting factors, including the 18S rRNA methyltransferase Bud23. Consistent with the known mechanisms of activation of methyltransferases by Trm112, we found that Trm112 interacts directly with Bud23 in vitro and that it is required for its stability in vivo. Consequently, trm112Δ cells are deficient for Bud23-mediated 18S rRNA methylation at position G1575 and for small ribosome subunit formation. Bud23 failure to bind nascent preribosomes activates a nucleolar surveillance pathway involving the TRAMP complexes, leading to preribosome degradation. Trm112 is thus active in rRNA, tRNA, and translation factor modification, ideally placing it at the interface between ribosome synthesis and function.
ETOC: Ribosome synthesis is a multistep process initiated in the nucleolus with the transcription of a precursor rRNA that is subjected to a series of modification and processing steps to generate the mature rRNA. In this paper, we describe a novel 60S ribosome biogenesis complex associating with LAS1L that controls rRNA processing and synthesis of the 28S rRNA.
The coordination of RNA polymerase I transcription with pre-rRNA processing, preribosomal particle assembly, and nuclear export is a finely tuned process requiring the concerted actions of a number of accessory factors. However, the exact functions of some of these proteins and how they assemble in subcomplexes remain poorly defined. LAS1L was first described as a nucleolar protein required for maturation of the 60S preribosomal subunit. In this paper, we demonstrate that LAS1L interacts with PELP1, TEX10, and WDR18, the mammalian homologues of the budding yeast Rix1 complex, along with NOL9 and SENP3, to form a novel nucleolar complex that cofractionates with the 60S preribosomal subunit. Depletion of LAS1L-associated proteins results in a p53-dependent G1 arrest and leads to defects in processing of the pre-rRNA internal transcribed spacer 2 region. We further show that the nucleolar localization of this complex requires active RNA polymerase I transcription and the small ubiquitin-like modifier–specific protease SENP3. Taken together, our data identify a novel mammalian complex required for 60S ribosomal subunit synthesis, providing further insight into the intricate, yet poorly described, process of ribosome biogenesis in higher eukaryotes.
We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation on Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.
The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.
Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Mitochondria perform a number of essential functions in the cell, including synthesis of ATP via the oxidative phosphorylation (OXPHOS) system. Normal mitochondrial function requires coordinated expression of two genomes: mitochondria's own genome (mtDNA), which encodes 13 respiratory chain subunits with essential structural and functional roles for the OXPHOS system, and the nuclear genome encoding the remaining ∼80 subunits. The mtDNA-encoded polypeptides are synthesized on mitochondrial ribosomes (mitoribosomes) located in the mitochondrial matrix. Biogenesis, maintenance and regulation of the complex mitochondrial translation apparatus are poorly understood despite its fundamental importance for cellular energy homeostasis. Here, we show that inactivation of the Nsun4 gene, encoding a mitochondrial m5C-methyltransferase, causes embryonic lethality, whereas tissue-specific disruption of Nsun4 in the heart causes cardiomyopathy with mitochondrial dysfunction. By performing sequencing of bisulfite-treated RNA we report that NSUN4 methylates C911 in 12S rRNA of the small ribosomal subunit. Surprisingly, NSUN4 can on its own perform this rRNA modification, whereas interaction with its partner protein MTERF4 is required for assembly of functional ribosomes. NSUN4 thus has dual roles in ribosome maturation and performs an important final quality control step to ensure that only mature mitoribosomal subunits are assembled into functional ribosomes.
The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C2H2-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.
Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome’s function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the indigenous methyltransferase RlmN and its evolutionary-related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily, and contain the characteristic cysteine rich CX3CX2C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-adenosylmethionine (SAM) is both the methyl donor and the source of a 5′-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully-assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90–92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in acquisition of antibiotic resistance.
RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m3U2634 and m3U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold–like methyltransferases and the point mutations in the S-adenosyl-l-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m5U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.
While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits (SSUs) and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of E. coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase deficient form of this protein. Our data reveals cold sensitivity and altered ribosomal profiles are associated with a ΔksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis prompting discussion of KsgA as a possible anti-microbial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.
KsgA; Dim1p; rRNA processing; ribosome biogenesis
Human ribosomal RNA processing is initiated by endonuclease cleavage and followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome.
Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference–mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.
A genome-wide screen of 4168 homozygous diploid yeast deletion
strains has been performed to identify nonessential genes that
participate in the bipolar budding pattern. By examining bud scar
patterns representing the sites of previous cell divisions, 127 mutants
representing three different phenotypes were found: unipolar,
axial-like, and random. From this screen, 11 functional classes of
known genes were identified, including those involved in
actin-cytoskeleton organization, general bud site selection, cell
polarity, vesicular transport, cell wall synthesis, protein
modification, transcription, nuclear function, translation, and other
functions. Four characterized genes that were not known previously to
participate in bud site selection were also found to be important for
the haploid axial budding pattern. In addition to known genes, we found
22 novel genes (20 are designated BUD13-BUD32) important
for bud site selection. Deletion of one resulted in unipolar budding
exclusively from the proximal pole, suggesting that this gene plays an
important role in diploid distal budding. Mutations in 20 other novel
BUD genes produced a random budding phenotype and one
produced an axial-like budding defect. Several of the novel Bud
proteins were fused to green fluorescence protein; two proteins were
found to localize to sites of polarized cell growth (i.e., the bud tip
in small budded cells and the neck in cells undergoing cytokinesis),
similar to that postulated for the bipolar signals and proteins that
target cell division site tags to their proper location in the cell.
Four others localized to the nucleus, suggesting that they play a role
in gene expression. The bipolar distal marker Bud8 was localized in a
number of mutants; many showed an altered Bud8-green fluorescence
protein localization pattern. Through the genome-wide identification
and analysis of different mutants involved in bipolar bud site
selection, an integrated pathway for this process is presented in which
proximal and distal bud site selection tags are synthesized and
localized at their appropriate poles, thereby directing growth at those
sites. Genome-wide screens of defined collections of mutants hold
significant promise for dissecting many biological processes in yeast.
The human WBSCR22 protein was previously shown to be up-regulated in invasive breast cancer and its ectopic expression enhances tumor cell survival in the vasculature. In the current study, we show that the WBSCR22 protein is important for cell growth. Knock-down of WBSCR22 with siRNA results in slower growth of WBSCR22-depleted cells. Treatment with siWBSCR22 causes defects in the processing of pre-rRNAs and reduces the level of free 40S ribosomal subunit, suggesting that WBSCR22 is involved in ribosome small subunit biosynthesis. The human WBSCR22 partially complements the growth of WBSCR22 yeast homologue, bud23 deletion mutant suggesting that the human WBSCR22 is a functional homologue of yeast Bud23. WBSCR22 is localized throughout the cell nucleus and is not stably associated with ribosomal subunits within the cell nucleus. We also show that the WBSCR22 protein level is decreased in lymphoblastoid cell lines derived from William-Beuren Syndrome (WBS) patients compared to healthy controls. Our data suggest that the WBSCR22 protein is a ribosome biogenesis factor involved in the biosynthesis of 40S ribosomal particles in mammalian cells.
Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA2 pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A2 within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.
Several mutants ( spb1 - spb7 ) have been previously identified as cold-sensitive extragenic suppressors of loss-of-function mutations in the poly(A)(+)-binding protein 1 of Saccharomyces cerevisiae. Cloning, sequence and disruption analyses revealed that SPB1 (YCL054W) encodes an essential putative S -adenosylmethionine-dependent methyltransferase. Polysome analyses showed an under-accumulation of 60S ribosomal subunits in the spb1-1 mutant and in a strain genetically depleted of Spb1p. Northern and primer extension analyses indicated that this was due to inhibition of processing of the 27SB precursors, which results in depletion of the mature 25S and 5.8S rRNAs. At later time points of Spb1p depletion, the stability of 40S ribosomal subunits is also affected. These results suggest that Spb1p is involved in 60S ribosomal subunit biogenesis and associates early with the pre-ribosomes. Consistent with this, hemagglutinin epitope-tagged Spb1p localizes to the nucleus with nucleolar enrichment. Despite the expected methyltransferase activity of Spb1p, global methylation of pre-rRNA is not affected upon Spb1p depletion. We propose that Spb1p is required for proper assembly of pre-ribosomal particles during the biogenesis of 60S ribosomal subunits.
Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m1A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m1A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.
The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23Δ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23Δ. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ∼45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function.
Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.
Gene networks are an efficient route for associating candidate genes with biological processes. Here, networks are used to discover more than 15 new genes for ribosomal subunit maturation, rRNA processing, and ribosomal export from the nucleus.
Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.
Ribosomes are the extremely complex cellular machines responsible for constructing new proteins. In eukaryotic cells, such as yeast, each ribosome contains more than 80 protein or RNA components. These complex machines must themselves be assembled by an even more complex machinery spanning multiple cellular compartments and involving perhaps 200 components in an ordered series of processing events, resulting in delivery of the two halves of the mature ribosome, the 40S and 60S components, to the cytoplasm. The ribosome biogenesis machinery has been only partially characterized, and many lines of evidence suggest that there are additional components that are still unknown. We employed an emerging computational technique called network-guided genetics to identify new candidate genes for this pathway. We then tested the candidates in a battery of experimental assays to determine what roles the genes might play in the biogenesis of ribosomes. This approach proved an efficient route to the discovery of new genes involved in ribosome biogenesis, significantly extending our understanding of a universally conserved eukaryotic process.
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.
During the synthesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms involved in the nuclear export of these particles and its coordination with other steps of the 40S synthesis pathway are mostly unknown. In this work we studied the function of Rrp12, the only major non-ribosomal factor of nuclear pre-40S particles that does not remain stably associated to them during maturation in the cytoplasm. We demonstrate that Rrp12 is required for the exit of pre-40S particles to the cytoplasm. Remarkably, we also found that Rrp12, together with the Crm1 exportin, participates in processes that occur in early pre-ribosomes in the nucleolus, including the processing of the pre-rRNA and the elimination of processing byproducts. Thus, Rrp12 and Crm1 participate in maturation steps that take place upstream of nuclear export. 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 nucleolar pre-ribosomes.
Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. By exploiting a combination of genetic, cell-biological, and biochemical approaches, we uncovered an unanticipated role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes. We show that recruitment of Mex67-Mtr2 to pre40S subunits requires loops emanating from its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops also recruit Mex67-Mtr2 to pre60S subunits and to the Nup84 complex, the respective interactions crucial for nuclear export of pre60S subunits and mRNAs. Thus Mex67-Mtr2 is a unique transport receptor that employs a common interaction surface to participate in the nuclear export of both pre-ribosomal subunits and mRNAs. Mex67-Mtr2 could engage a regulatory crosstalk among the three major export pathways for optimal cellular growth and proliferation.
Eukaryotic pre-ribosomal subunits (pre40S and pre60S) are one of the largest RNA containing cargos that are transported from the nucleus through the nuclear pore complex (NPC) into the cytoplasm. While genetic approaches in budding yeast have identified bona fide export factors for pre60S subunits, little is known regarding nuclear export of pre40S subunits. The conserved transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus, and facilitates their passage through the NPC, by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel. Here, we report an additional role of Mex67-Mtr2. We show that Mex67-Mtr2 binds pre40S subunits via loops present on its NTF2-like domains and that the C-terminal FG-rich nucleoporin interacting UBA-like domain within Mex67 contributes to the transport of pre40S subunits to the cytoplasm. Remarkably, the same loops of Mex67-Mtr2 also contribute to the nuclear export of pre60S subunits and mRNAs. Thus, the transport receptor Mex67-Mtr2 could employ a common interaction surface to engage a regulatory crosstalk among the three major export pathways.
A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes.
The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.
Ribosomes are molecular machines that synthesize proteins and are found in every cell. Each ribosome has a small and a large subunit, each of which is in turn composed of ribosomal RNA and many ribosomal proteins. In eukaryotic cells, the generation of these ribosomal subunits is a complex process requiring the participation of hundreds of factors. Originating from the nucleolus (a nuclear region specialized in ribosome synthesis) immature ribosomal subunits pass through the nuclear interior and are exported to the cytoplasm, where their assembly is finalized. In recent years, it has become apparent that defects in ribosome production are associated with human diseases, but our knowledge about this fundamental process is largely based on knowledge derived from yeast, a unicellular eukaryote. We set out to systematically identify factors involved in making ribosomes in human cells by individually depleting around 500 different cellular proteins. Using microscopic analysis, we identified approximately 150 human ribosome synthesis factors, thereby significantly extending our knowledge about human ribosome biogenesis. Our dataset not only revealed many evolutionarily conserved aspects of this essential cellular process but also led us to characterize an export route for the large ribosomal subunit that is specific for higher eukaryotic cells.
The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA’s important role in ribosome biogenesis has been recently expanded in Escherichia coli; these studies help explain why KsgA is so highly conserved and also suggest KsgA’s potential as an antimicrobial drug target.
We have analyzed KsgA’s contribution to ribosome biogenesis and cell growth in Staphylococcus aureus. We found that deletion of ksgA in S. aureus led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in E. coli. Additionally, the ksgA knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature.
This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in S. aureus are in contrast to results previously described in E. coli, where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.
KsgA; Ribosome biogenesis; Staphylococcus aureus; Escherichia coli; Methyltransferase