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.     

Characterization of ribonucleoprotein subparticles from 50 S ribosomal subunits of Escherichia coli.

Large ribonucleoprotein subparticles were recovered upon ribonuclease digestion of the 50 S ribosomal subunits of Escherichia coli, partially deproteinized by LiCl. Both their RNA and their protein compositions were analysed. The subunits, treated with LiCl at a concentration of 5.5 m, released an homogeneous subparticle containing proteins L₃, L₄, L₁₃, L₁₇, L₂₂ and L₂₉, about 70% of the 13 S fragment of 23 S RNA and about 50% of the 18 S one. Slightly larger species of subparticles were obtained from 50 S subunits treated with LiCl at concentrations between 3 m and 5 m; they contained in addition proteins L₂₀, L₂₁ and L₂₃ or L₂, L₁₄, L₂₀, L₂₁ and L₂₃ and a few small 23 S RNA fragments. No large subparticle was recovered from the 6 m-LiCl-treated 50 S subunits which contain only proteins L₃, L₁₃ and L₁₇. These LiCl subparticles were compared with those obtained from intact, unfolded and sodium doecyl sulphatetreated 50 S subunits.These studies reveal that in the presence of 0.10 m-magnesium acetate there is a very compact area within 50 S subunits consisting of proteins L₃, L₄, L₁₃, L₁₇, L₂₂ and L₂₉ and of about 60% of 23 S RNA; this area probably has an essential structural role. The results also show that 23 S RNA has a more folded conformation when within the 50 S subunit than when isolated, this conformation being stabilized by some of the 50 S proteins, in particular proteins L₄, L₂₂, L₂₀ and L₂₁. Finally these data permit a more definite localization of the primary and/or secondary binding sites of proteins L₂, L₃, L₄, L₁₄, L₁₇, L₂₀, L₂₁ and L₂₂ on 23 S RNA.     

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.     

Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.     

Photochemical cross-linking of translation initiation factor 3 to Escherichia coli 50S ribosomal subunits

Translation initiation factor 3 (IF-3) was bound noncovalently to Escherichia coli 50S ribosomal subunits. Irradiation of such complexes with near-ultraviolet light (greater than 285 nm) resulted in covalent attachment of initiation factor 3 to the 50S subunit. Photo-cross-linking attained its maximum level of 40% of that which was noncovalently bound after 90 min of irradiation. Cross-linking was abolished in the presence of either 0.5 M NH4C1 or 0.25 mM aurintricarboxylic acid, indicating that specific binding of initiation factor 3 to the ribosome was a prerequisite for subsequent covalent attachment. Further analysis showed that all the IF-3 was covalently bound to a small number of 50S subunit proteins. The major cross-linked proteins were identified as L2, L7/L12, L11, and L27 by immunochemical techniques. These results are discussed in light of the proposed mechanism for IF-3 function.     

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.     

A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes

The rate constants for eucaryotic initiation factor 3 (eIF3) association and dissociation with 40S ribosomal subunits and 80S monosomes have been determined. These rate constants were determined by laser light scattering with unmodified eIF3. The affinity of eIF3 for 40S subunits is about 30-fold greater than for 80S ribosomes. This difference in affinity resides mainly in the association rate constants. Rate constants of 8.8 X 10(7) and 7.3 X 10(6) M-1 s-1 were obtained for eIF3 binding to 40S subunits and 80S ribosomes, respectively. From thermodynamic cycles, the affinity of eIF3-40S subunits for 60S subunits is about 30-fold lower than free 40S subunits for 60S subunits. A calculation shows that under these conditions and assuming simple equilibria, approximately 12% of ribosomal subunits would associate via a reaction of 40S-eIF3 with 60S subunits as opposed to a path where eIF3 dissociates from the 40S subunits prior to association with 60S subunits.     

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j-, respectively). Although unlike eIF3j+, eIF3j- does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2*GTP*Met-tRNA(i)Met ternary complex, eIF3j-/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j- nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.     

Structural and functional studies of ribonucleoprotein fragments isolated from Escherichia coli 50 S ribosomal subunits.

Ribonuclease digestion of 50 S-derived LiCl cores led to 22 ribonucleoprotein particles which were isolated by repeated sucrose gradient centrifugations. The protein content was determined and ranged from 2 to 28 proteins. Most of the fragments showed a unique RNA pattern as judged by acrylamide gel electrophoresis.Functional tests were performed with selected fragments. No fragment was active in the poly(U) or the peptidyl-transferase assay. Chloramphenicol binding studies revealed that in addition to the dominant role of protein L16, the protein L11 (or L6) is involved directly in the drug binding. Finally, tests for ATPase and GTPase activity showed that protein L18 is involved in GTPase activity.     

Spatial proximity of the ribosomal mRNA binding center to the 50S subunit]

4-(N-2-chloroethyl-N-methylamino)benzylamide of 5'-heptaadenylic acid was used for affinity labelling of the ribosome in the vicinity of its mRNA-binding centre. This derivative, similar to the free oligonucleotide, stimulates the binding of [14C]-lysyl-tRNA to ribosomes of E. coli and alkylates ribosomes both the 30S and the 50S subunits. The alkylation of ribosomes is inhibited by pre-incubation of ribosomes with polyadenylic acid, which suggests that the chemical modification is a specific one and occurs in the vicinity of mRNA-binding site. The fact, that a short oligonucleotide having an active group on its 5'end attacks the 50S subunit of ribosome may indicate that the mRNA-binding centre is located in the contact region between ribosomal subunits.     

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.     

Covalent attachment of poly (U) template to 40S - mammalian ribosomal subunits

When 40S subunits are irradiated at 254nm in presence of [³H] poly (U), formation of a 40S subunit-poly (U) complex can be demonstrated either by filtration technique at low Mg⁺⁺ concentration or by polyacrylamide gel electrophoresis. No stable complex was detected using unirradiated samples under the same conditions. Electrophoresis of this complex in the presence of dodecyl sulfate showed that part of the poly (U) directly associates with 18S RNA. This association is not through proteins, since it is not disrupted by pronase treatment.     

The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.     

GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.

Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.     

Binding of globin mRNA, beta-globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein

The binding of rabbit globin mRNA, in-vitro-generated beta-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of beta-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free beta-globin mRNA. A protein of Mr 90,000 binds specifically the 3'-nontranslated trailer of the poly(A)-free beta-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72,000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.