Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

The course of the assembly of ribosomal subunits in yeast

The course of the assembly of the various ribosomal proteins of yeast into ribosomal particles has been studied by following the incorporation of radioactive individual protein species in cytoplasmic ribosomal particles after pulse-labelling of yeast protoplasts with tritiated amino acids. The pool of ribosomal proteins is small relative to the rate of ribosomal protein synthesis, and, therefore, does not affect essentially the appearance of labelled ribosomal proteins on the ribosomal particles. From the labelling kinetics of individual protein species it can be concluded that a number of ribosomal proteins of the 60 S subunit (L6, L7, L8, L9, L11, L15, L16, L23, L24, L30, L32, L36, L40, L41, L42, L44 and L45) associate with the ribonucleoprotein particles at a relatively late stage of the ribosomal maturation process. The same was found to be true for a number of proteins of the 40 S ribosomal subunit (S10, S27, S31, S32, S33 and S34). Several members (L7, L9, L24 and L30) of the late associating group of 60-S subunit proteins were found to be absent from a nuclear 66 S precursor ribosomal fraction. These results indicate that incorporation of these proteins into the ribosomal particles takes place in the cytoplasm at a late stage of the ribosomal maturation process.     

Mapping the three-dimensional locations of ribosomal RNA and proteins.

Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory. In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy. The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA. A novel structure, the platform ring, is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site. In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory. In order to evaluate the reliability of our model for the three-dimensional distribution of 16S rRNA, we have predicted which sites of rRNA are adjacent to ribosomal proteins and compared these predictions with r-protein protection studies of others. Good correlation between the model, the locations of rRNA sites, the locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins, provides independent support for this model.     

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.     

Cross-linking of Met-tRNAf to eIF-2 beta and to the ribosomal proteins S3a and S6 within the eukaryotic inhibition complex, eIF-2 .GMPPCP.Met-tRNAf.small ribosomal subunit.

In the quaternary initiation complex, eIF-2.GMPPCP.Met-tRNAf.40S ribosomal subunit, the Met-tRNAf can be cross-linked to the beta subunit of initiation factor eIF-2 as well as to ribosomal proteins S3a and S6 by treatment with the bifunctional reagent, diepoxybutane. Using 40S subunits, modified in advance with the heterobifunctional reagent, methyl-rho-azido-benzoylaminoacetimidate, Met-tRNAf is covalently bound to the same ribosomal proteins (S3a and S6) upon irradiation of the complex with ultraviolet light. Under both conditions proteins S3a and S6, together with a limited number of other ribosomal proteins, are covalently bound to 18S ribosomal RNA.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.     

The spatial arrangement of the complex between eukaryotic initiation factor eIF-3 and 40 S ribosomal subunit. Cross-linking between factor and ribosomal proteins

The binding site for eIF-3 on the small ribosomal subunit was studied (a) by use of a complex of eIF-3 and derived 40 S ribosomal subunit from rat liver, and (b) by use of native small ribosomal subunits from rabbit reticulocytes. After treatment of both complexes with dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate ribosomal proteins S3a, S4, S6, S7, S8, S9, S10, S23/24 and S27 became covalently linked to eIF-3 and were isolated together with the factor by gradient centrifugation. The ribosomal proteins were identified by two-dimensional polyacrylamide gel electrophoresis after periodate cleavage of the link(s).     

Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

The protein composition of HeLa ribosomal subunits and nucleolar precursor particles

Using two-dimensional polyacrylamide gel electrophoresis, the protein patterns from HeLa 80S and 55S nucleolar precursor particles have been compared with those of cytoplasmic 40S and 60S ribosomal subunits. The 55S particle was found to have 21 anionic and 52 cationic proteins, including 18 large subunit ribosomal proteins. The 80S precursor pattern was identical to the 55S pattern except three anionic and four cationic proteins were absent. Of those missing cations, three were large subunit proteins. However, no small subunit ribosomal proteins were detected on either precursor. Numerous high molecular weight non-ribosomal proteins were found in both precursor particles and may correspond to a class of stable nucleolar proteins.     

The topography of ribosomal proteins on the surface of the 30S subunit of Escherichia coli

Eight ribosomal proteins, S6, S10, S11, S15, S16, S18, S19 and S21 have been localized on the surface of the 30S subunit from Escherichia coli by immuno electron microscopy. The specificity of the antibody binding sites was demonstrated by stringent absorption experiments. In addition we have reinvestigated and refined the locations of proteins S5, S13 and S14 on the ribosomal surface which had previously been localized in our laboratory (Tischendorf et al., Mol. Gen. Genet. 134, 209-223, 1974). Thus altogether 16 out of the 21 ribosomal proteins of the small subunit from E. coli have been mapped in our laboratory.     

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.     

Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.     

Modification of yeast ribosomal proteins. Phosphorylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3(mt) binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3(mt) cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3(mt) has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3(mt) except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.