Nuclear export of ribosomal subunits

The partitioning of cells by a nuclear envelope ensures that precursors of ribosomes do not interact prematurely with other components of the translation machinery. Ribosomal subunits are assembled in nucleoli and exported to the cytoplasm in a CRM1/Ran-GTP-dependent fashion. Export of the large (60S) subunit requires a shuttling adaptor protein, NMD3, which binds to mature, correctly folded subunits. Immature or defective particles do not bind NMD3 and thus are excluded from the export pathway. This structural proofreading is extended into the cytoplasm, where it is believed that several energy-requiring steps release shuttling factors from the subunit, allowing it to function in translation.     

Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells

It is generally assumed that, in mammalian cells, preribosomal RNAs are entirely processed before nuclear exit. Here, we show that pre-40S particles exported to the cytoplasm in HeLa cells contain 18S rRNA extended at the 3' end with 20-30 nucleotides of the internal transcribed spacer 1. Maturation of this pre-18S rRNA (which we named 18S-E) involves a cytoplasmic protein, the human homolog of the yeast kinase Rio2p, and appears to be required for the translation competence of the 40S subunit. By tracking the nuclear exit of this precursor, we have identified the ribosomal protein Rps15 as a determinant of preribosomal nuclear export in human cells. Interestingly, inhibition of exportin Crm1/Xpo1 with leptomycin B strongly alters processing of the 5'-external transcribed spacer, upstream of nuclear export, and reveals a new cleavage site in this transcribed spacer. Completion of the maturation of the 18S rRNA in the cytoplasm, a feature thought to be unique to yeast, may prevent pre-40S particles from initiating translation with pre-mRNAs in eukaryotic cells. It also allows new strategies for the study of preribosomal transport in mammalian cells.     

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.     

Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2

The yeast Mex67-Mtr2 complex and its homologous metazoan counterpart TAP-p15 operate as nuclear export receptors by binding and translocating mRNA through the nuclear pore complexes. Here, we show how Mex67-Mtr2 can also function in the nuclear export of the ribosomal 60S subunit. Biochemical and genetic studies reveal a previously unrecognized interaction surface on the NTF2-like scaffold of the Mex67-Mtr2 heterodimer, which in vivo binds to pre-60S particles and in vitro can interact with 5S rRNA. Crucial structural requirements for this binding platform are loop insertions in the middle domain of Mex67 and Mtr2, which are absent from human TAP-p15. Notably, when the positively charged amino acids in the Mex67 loop are mutated, interaction of Mex67-Mtr2 with pre-60S particles and 5S rRNA is inhibited, and 60S subunits, but not mRNA, accumulate in the nucleus. Thus, the general mRNA exporter Mex67-Mtr2 contains a distinct electrostatic interaction surface for transporting 60S preribosomal cargo.     

Incorporation of the yeast mitochondrial ribosomal protein Mrp2 into ribosomal subunits requires the mitochondrially encoded Var1 protein

Mrp2 is a protein component of the small subunit of mitochondrial ribosomes in the yeast Saccharomyces cerevisiae. We have examined the expression of Mrp2 in yeast mutants lacking mitochondrial DNA and found that the steady-state level of Mrp2 is dramatically decreased relative to wild type. These data suggest that the accumulation of Mrp2 depends on the expression of one or more mitochondrial gene products. The mitochondrial genome of S. cerevisiae encodes two components of the small ribosomal subunit, 15S rRNA and the Var1 protein, both of which are necessary for the formation of mature 37S subunits. Several studies have shown that in the absence of Var1 incomplete subunits accumulate, which lack a limited number of ribosomal proteins. Here, we show that Mrp2 is one of the proteins absent from subunits lacking Var1, indicating that Var1 plays an important role in the incorporation of Mrp2 into mitochondrial ribosomal subunits.     

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2. Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm.     

Nuclear export and cytoplasmic maturation of ribosomal subunits

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.     

Modification of 40S ribosomal subunits from yeast with dimethylmaleic anhydride

Modification of 40S ribosomal subunits from Saccharomyces cerevisiae with dimethylmaleic anhydride (DMMA), a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity and by dissociation of proteins from the particles. The protein-deficient ribosomal particles, originated from 40S subunits by treatment with dimethylmaleic anhydride at a molar ratio of reagent to particle of 250, can partially reconstitute active subunits upon addition of the corresponding released proteins, and regeneration of the modified amino groups.

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Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal

Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA. Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain. These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis. Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion [Chapman, N.M., & Noller, H.F. (1977) J. Mol. Biol 109, 131-149] that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement. Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation.     

A Noc complex specifically involved in the formation and nuclear export of ribosomal 40 S subunits

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A(0), A(1), and A(2). Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.     

Two-step purification of the major phosphorylated protein in reticulocyte 40S ribosomal subunits

In reticulocytes, a single ribosomal protein, S13, has been shown to be phosphorylated by the cAMP-regulated protein kinases. The 40S ribosomal subunits were phosphorylated in vitro with [gamma-32P]ATP to facilitate the identification of S13 during the two-step purification procedure. Total ribosomal protein from the 40S subunit was fractionated by phosphocellulose chromatography in urea, and S13 was purified to homogeneity by gel filtration on Sephadex G-100. The protein was identified by the radioactive phosphate, by molecular weight, and by the migration characteristics in a two-dimensional polyacrylamide gel electrophoresis system. Thin-layer electrophoresis of partial acid hydrolysates of S13 showed that more than one phosphorylated residue was present in the same oligopeptide, indicating at least some of the phosphoryl groups were clustered in the protein molecule.     

Disassembly and reconstitution of yeast 60S ribosomal subunits

Summary Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins. The reconstituted particles can be isolated by centrifugation without loss of activity, having the protein composition of the original subunits.Abbreviations DMMA Dimethylmaleic Anhydride     

Partial reconstitution of 60S ribosomal subunits from yeast

Summary Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20–40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH₄Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH₄Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.Abbreviation DMMA dimethylmaleic anhydride     

Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits

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 bud23Delta 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.     

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins. Upf1 coimmunoprecipitates specifically with epitope-tagged 40S ribosomal subunits, and Upf1 association with high-salt washed or puromycin-released 40S subunits was found to occur without simultaneous eRF1, eRF3, Upf2, or Upf3 association. Two-hybrid analyses and in vitro binding assays identified a specific interaction between Upf1 and Rps26. Using mutations in domains of UPF1 known to be crucial for its function, we found that Upf1:40S association is modulated by ATP, and Upf1:Rps26 interaction is dependent on the N-terminal Upf1 CH domain. The specific association of Upf1 with the 40S subunit is consistent with the notion that this RNA helicase not only triggers rapid decay of nonsense-containing mRNAs, but may also have an important role in dissociation of the premature termination complex.     

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.     

Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits

Our knowledge of the functions of metazoan ribosomal proteins in ribosome synthesis remains fragmentary. Using siRNAs, we show that knockdown of 31 of the 32 ribosomal proteins of the human 40S subunit (ribosomal protein of the small subunit [RPS]) strongly affects pre–ribosomal RNA (rRNA) processing, which often correlates with nucleolar chromatin disorganization. 16 RPSs are strictly required for initiating processing of the sequences flanking the 18S rRNA in the pre-rRNA except at the metazoan-specific early cleavage site. The remaining 16 proteins are necessary for progression of the nuclear and cytoplasmic maturation steps and for nuclear export. Distribution of these two subsets of RPSs in the 40S subunit structure argues for a tight dependence of pre-rRNA processing initiation on the folding of both the body and the head of the forming subunit. Interestingly, the functional dichotomy of RPS proteins reported in this study is correlated with the mutation frequency of RPS genes in Diamond-Blackfan anemia.     

Linear ubiquitin fusion to Rps31 and its subsequent cleavage are required for the efficient production and functional integrity of 40S ribosomal subunits

The post-translational modifier ubiquitin is generated exclusively by proteolytic cleavage of precursor proteins. In Saccharomyces cerevisiae , cleavage of the linear precursor proteins releases ubiquitin and the C-terminally fused ribosomal proteins Rpl40 (Ubi1/2 precursor) and Rps31 (Ubi3 precursor), which are part of mature 60S and 40S ribosomal subunits respectively. In this study, we analysed the effects of ubi3 mutations that interfere with cleavage of the ubiquitin–Rps31 fusion protein. Strikingly, the lethal ubi3 + P77 mutation, which abolished cleavage almost completely, led to a rapid G1 cell cycle arrest upon genetic depletion of wild-type UBI3 . Under these conditions, the otherwise unstable Ubi3+P77 protein was efficiently assembled into translation-competent 40S ribosomal subunits. In contrast to the cleavage-affecting mutations, deletion of the ubiquitin moiety from UBI3 led to a decrease in 40S ribosomal subunits and to the incorporation of the 20S pre-rRNA into polyribosomes. Altogether, our findings provide additional evidence that the initial presence of the ubiquitin moiety of Ubi3 contributes to the efficient production of 40S ribosomal subunits and they suggest that ubiquitin release is a prerequisite for their functional integrity.