PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-18 (18)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Physical and Functional Interaction between the Methyltransferase Bud23 and the Essential DEAH-Box RNA Helicase Ecm16 
Molecular and Cellular Biology  2014;34(12):2208-2220.
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.
doi:10.1128/MCB.01656-13
PMCID: PMC4054285  PMID: 24710271
2.  Integrity of the P-site is probed during maturation of the 60S ribosomal subunit 
The Journal of Cell Biology  2012;197(6):747-759.
The P-site of the 60S ribosomal subunit signals to Tif6 via Elf1 during ribosomal maturation, suggesting a quasifunctional check of the integrity of the 60S subunit before the first round of translation.
Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 Å away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S subunit before its first round of translation.
doi:10.1083/jcb.201112131
PMCID: PMC3373404  PMID: 22689654
3.  Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit 
The Journal of Cell Biology  2010;189(7):1079-1086.
3D reconstruction by cryo-EM provides the first structural description of a ribosomal biogenesis factor (Nmd3) in complex with the 60S ribosomal subunit.
The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.
doi:10.1083/jcb.201001124
PMCID: PMC2894450  PMID: 20584915
4.  Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0 
The Journal of Cell Biology  2009;186(6):849-862.
The step by step assembly process from preribosome in the nucleus to translation-competent 60S ribosome subunit in the cytoplasm is revealed (also see Kemmler et al. in this issue).
The ribosome stalk is essential for recruitment of translation factors. In yeast, P0 and Rpl12 correspond to bacterial L10 and L11 and form the stalk base of mature ribosomes, whereas Mrt4 is a nuclear paralogue of P0. In this study, we show that the dual-specificity phosphatase Yvh1 is required for the release of Mrt4 from the pre-60S subunits. Deletion of YVH1 leads to the persistence of Mrt4 on pre-60S subunits in the cytoplasm. A mutation in Mrt4 at the protein–RNA interface bypasses the requirement for Yvh1. Pre-60S subunits associated with Yvh1 contain Rpl12 but lack both Mrt4 and P0. These results suggest a linear series of events in which Yvh1 binds to the pre-60S subunit to displace Mrt4. Subsequently, P0 loads onto the subunit to assemble the mature stalk, and Yvh1 is released. The initial assembly of the ribosome with Mrt4 may provide functional compartmentalization of ribosome assembly in addition to the spatial separation afforded by the nuclear envelope.
doi:10.1083/jcb.200904110
PMCID: PMC2753163  PMID: 19797078
5.  Reengineering Ribosome Export 
Molecular Biology of the Cell  2009;20(5):1545-1554.
Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.
doi:10.1091/mbc.E08-10-1000
PMCID: PMC2649259  PMID: 19144820
6.  Eukaryotic rpL10 drives ribosomal rotation 
Nucleic Acids Research  2013;42(3):2049-2063.
Ribosomes transit between two conformational states, non-rotated and rotated, through the elongation cycle. Here, we present evidence that an internal loop in the essential yeast ribosomal protein rpL10 is a central controller of this process. Mutations in this loop promote opposing effects on the natural equilibrium between these two extreme conformational states. rRNA chemical modification analyses reveals allosteric interactions involved in coordinating intersubunit rotation originating from rpL10 in the core of the large subunit (LSU) through both subunits, linking all the functional centers of the ribosome. Mutations promoting rotational disequilibria showed catalytic, biochemical and translational fidelity defects. An rpL3 mutation promoting opposing structural and biochemical effects, suppressed an rpL10 mutant, re-establishing rotational equilibrium. The rpL10 loop is also involved in Sdo1p recruitment, suggesting that rotational status is important for ensuring late-stage maturation of the LSU, supporting a model in which pre-60S subunits undergo a ‘test drive’ before final maturation.
doi:10.1093/nar/gkt1107
PMCID: PMC3919601  PMID: 24214990
7.  The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits 
Molecular Biology of the Cell  2012;23(21):4313-4322.
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.
doi:10.1091/mbc.E12-05-0370
PMCID: PMC3484107  PMID: 22956767
8.  Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae 
Nucleic Acids Research  2012;41(2):1135-1150.
Ribosome biogenesis is a multi-step process that couples cell growth with cell proliferation. Although several large-scale analysis of pre-ribosomal particles have identified numerous trans-acting factors involved in this process, many proteins involved in pre-rRNA processing and ribosomal subunit maturation have yet to be identified. Las1 was originally identified in Saccharomyces cerevisiae as a protein involved in cell morphogenesis. We previously demonstrated that the human homolog, Las1L, is required for efficient ITS2 rRNA processing and synthesis of the 60S ribosomal subunit. Here, we report that the functions of Las1 in ribosome biogenesis are also conserved in S. cerevisiae. Depletion of Las1 led to the accumulation of both the 27S and 7S rRNA intermediates and impaired the synthesis of the 60S subunit. We show that Las1 co-precipitates mainly with the 27S rRNA and associates with an Nsa1 and Rix1-containing pre-60S particle. We further identify Grc3 as a major Las1-interacting protein. We demonstrate that the kinase activity of Grc3 is required for efficient pre-rRNA processing and that depletion of Grc3 leads to rRNA processing defects similar to the ones observed in Las1-depleted cells. We propose that Las1 and Grc3 function together in a conserved mechanism to modulate rRNA processing and eukaryotic ribosome biogenesis.
doi:10.1093/nar/gks1086
PMCID: PMC3553937  PMID: 23175604
9.  Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit 
Molecular cell  2010;39(2):196-208.
In eukaryotic cells the final maturation of ribosomes occurs in the cytoplasm, where trans-acting factors are removed and critical ribosomal proteins are added for functionality. Here, we have carried out a comprehensive analysis of cytoplasmic maturation, ordering the known steps into a coherent pathway. Maturation is initiated by the ATPase Drg1. Downstream, assembly of the ribosome stalk is essential for the release of Tif6. The stalk recruits GTPases during translation. Because the GTPase Efl1, which is required for the release of Tif6, resembles the translation elongation factor eEF2, we suggest that assembly of the stalk recruits Efl1, triggering a step in 60S biogenesis that mimics aspects of translocation. Efl1 could thereby provide a mechanism to functionally check the nascent subunit. Finally, the release of Tif6 is a prerequisite for the release of the nuclear export adapter Nmd3. Establishing this pathway provides an important conceptual framework for understanding ribosome maturation.
doi:10.1016/j.molcel.2010.06.018
PMCID: PMC2925414  PMID: 20670889
ribosome; ribosome biogenesis; EFL1; NMD3; TIF6
10.  Maturation of Eukaryotic Ribosomes: Acquisition of Functionality 
Trends in biochemical sciences  2010;35(5):260-266.
In eukaryotic cells ribosomes are preassembled in the nucleus and exported to the cytoplasm where they undergo final maturation. This involves the release of trans-acting shuttling factors, transport factors, incorporation of the remaining ribosomal proteins and final rRNA processing steps. Recent work, especially on the large (60S) ribosomal subunit, has made it abundantly clear that the 60S subunit is exported from the nucleus in a functionally inactive state. Its arrival in the cytoplasm triggers events that that render it translationally competent. Here we focus on these cytoplasmic maturation events and speculate about why eukaryotic cells have evolved such an elaborate pathway of maturation.
doi:10.1016/j.tibs.2010.01.001
PMCID: PMC2866757  PMID: 20137954
11.  Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics 
PLoS Biology  2009;7(10):e1000213.
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.
Author Summary
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.
doi:10.1371/journal.pbio.1000213
PMCID: PMC2749941  PMID: 19806183
12.  Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits▿  
Molecular and Cellular Biology  2008;28(10):3151-3161.
BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.
doi:10.1128/MCB.01674-07
PMCID: PMC2423152  PMID: 18332120
13.  Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast 
Molecular Biology of the Cell  2008;19(2):735-744.
We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.
doi:10.1091/mbc.E07-09-0968
PMCID: PMC2230582  PMID: 18077551
14.  Nuclear Recycling of the Pre-60S Ribosomal Subunit-Associated Factor Arx1 Depends on Rei1 in Saccharomyces cerevisiae 
Molecular and Cellular Biology  2006;26(10):3718-3727.
Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1Δ mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1Δ mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1Δ mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1Δ cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.
doi:10.1128/MCB.26.10.3718-3727.2006
PMCID: PMC1489010  PMID: 16648468
15.  Defining the Order in Which Nmd3p and Rpl10p Load onto Nascent 60S Ribosomal Subunits 
Molecular and Cellular Biology  2005;25(9):3802-3813.
The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.
doi:10.1128/MCB.25.9.3802-3813.2005
PMCID: PMC1084314  PMID: 15831484
16.  Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit 
The Journal of Cell Biology  2000;151(5):1057-1066.
In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.
PMCID: PMC2174350  PMID: 11086007
nuclear export; ribosome; Crm1p; Nmd3p; Saccharomyces cerevisiae
17.  Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p 
Molecular and Cellular Biology  2000;20(11):4006-4015.
The RAT1 gene of Saccharomyces cerevisiae encodes a 5′→3′ exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8SL was the predominant form of 5.8S and there was an accumulation of 3′-extended forms but not 5′-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5′ and 3′ processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.
PMCID: PMC85771  PMID: 10805743
18.  NMD3 Encodes an Essential Cytoplasmic Protein Required for Stable 60S Ribosomal Subunits in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(3):2389-2399.
A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx2C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.
PMCID: PMC84031  PMID: 10022925

Results 1-18 (18)