Permuted 23S rRNA is integrated in 50S ribosome particles in Thermococcus barophilus

In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase center essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S nonfunctional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.

In recent years, many research groups have focused their interest on 5S rRNA, the smallest RNA component of the ribosome that is preserved in almost all organisms. Its precise role in the ribosome, the protein “biosynthetic machine”, has not been fully clarified. The results of structural studies place 5S rRNA in the large ribosomal subunit. Findings concerning relations between structure and function lead to the assumption that 5S rRNA stabilises the structure of the ribosome and acts as natural transmitter of information between functional centres. On the other hand, the ionic environment directly affects ribosome function. Monovalent and divalent ions as well as polycations, such as polyamines, influence the ribosomal conformation, and therefore are involved in the function of the ribosome. Preliminary studies in our laboratory have shown that polyamines, namely spermine, stabilize the secondary and tertiary structure of 5S rRNA, leading to a more cohesive configuration of the molecule. The aim of this thesis was to investigate the role of 5S rRNA configuration in protein synthesis of the enterobacterium E.coli. At the same time, the biological activity of polyamines in the structure and functioning of both 5S rRNA and the whole ribosomal complex was studied. Polyamine binding to 5S rRNA was investigated by photoaffinity labeling, using a photoreactive analogue of spermine, ABA-spermine, as a molecular probe. Kinetic study was performed and the number of cross-linking sites of ABA-spermine in free 5S rRNA, or 5S rRNA incorporated into 50S subunits or 70S ribosomal complexes from E.coli ribosomes was determined. The results revealed that in free 5S rRNA spermine binds mainly to nucleotides of helices III and V and loops A, C, D and E. Integration of 5S rRNA into 50S subunits or 70S poly (U) – programmed ribosomes results in a great reduction of the susceptibility of 5S rRNA to ABA-spermine. The impact of the ABA-spermine photo-incorporation into the 5S rRNA molecule was then investigated. Chemical modification experiments revealed that, regardless of whether 5S rRNA is free or assembled in 50S subunits or 70S ribosomal complexes, binding of ABA-spermine causes significant changes in the conformation of the molecule. Loop A adopts a “looser” structure, while loops C, D, E and helices III and V achieve a more compact folding. With the discovery of changes in the configuration of 5S rRNA, due to ABA-spermine’s binding, it was interesting to study whether these conformational changes influence fundamental functions of the ribosome. Initially, the effect of changing the configuration of 5S rRNA on 50S subunit and 70S ribosomal complex assembly was investigated. The results showed that binding of ABA-spermine to 5S rRNA does not eliminate the process of 50S subunit assembly, but impedes the necessary structural changes by stabilising a more coherent structure of 5S rRNA, thermodynamically less favourable for the process. Despite the fact that binding of ABA-spermine to 5S rRNA results in structural changes that reduce the assembly of 50S subunits, when the later are formed they acquire such a structure that allows their interaction with 30S subunits. Nevertheless, the presence of 5S rRNA seems to be necessary for the formation of active 50S subunits, since 5S RNA-depleted ribosomal subunits do not associate with 30S subunits to functional 70S ribosomes. Next, the effect of photolabeling 5S rRNA was studied, initially on the ability of ribosomes to bind substrate, and then on their catalytic activity. Photolabeling of 5S rRNA with ABA-spermine caused a small but obvious activation in AcPhe-tRNA binding to the P-site. In contrast, binding to the A-site did not appear to be affected. Of great importance was the effect on the activity of peptidyl-transferase; in the presence of spermine, an increase of up to 30% on the activity of PTase was observed. Interestedly, ribosomes that lack 5S rRNA maintained ~ 10% of PTase activity, which leads to the assumption that 5S rRNA is not absolutely necessary for the formation of peptide bonds. The study of the effect of 5S rRNA conformational changes on ribosome's ability to translocate substrates followed. Spontaneous (non-enzymatic) translocation was studied first, and then, EF-G-catalyzed translocation. The results confirmed the assumption that translocation of substrates is an inherent property of the ribosome itself, even though extremely slow. At the same time, it was shown that binding of spermine to 5S rRNA, stimulates non-enzymatic translocation, possibly through changes in the folding of ribosomal RNA, that are beneficial in translocating tRNAs. Further, the presence of spermine in 5S rRNA reduced the requirements for EF-G. This result can be explained either by a possible beneficial effect of spermine to the binding of EF-G to the ribosome, either through its impact in the process of hydrolysis of GTP. To clarify which of the two versions is accurate, we studied the effect of 5S rRNA conformational changes on the ribosomal ability to bind EF-G, and then on their ability to trigger EF-G-dependent GTP hydrolysis. It was found that the presence of spermine in 5S rRNA promotes EF-G binding to ribosomes. In contrast, the results of the study showed that the initial rate of GTP hydrolysis was not influenced by the presence of spermine. Ribosomes depleted of 5S rRNA retained some GTP activity, but were unable to effectively bind EF-G even in the presence of spermine. Taking into account that EF-G binds in domain II and VI of 23S rRNA, but direct contacts of 5S rRNA with these areas or with EF-G have not been found, we are led to the conclusion that 5S rRNA affects EF-G-GTP binding to ribosomes, through allosteric signals, without affecting the EF-G-mediated GTP hydrolysis. To confirm the above, we studied the effect of the ionic environment and the changes in the tertiary structure of 5S rRNA on the conformational state of fundamental functional centres in the ribosome. Specifically, the study was focused on the ability of free spermine or photolabeling 5S rRNA with ABA-spermine to affect the SRL and GAC regions of the ribosome as well as the A-and P-sites. It was found that interaction of free spermine with ribosomes intensifies the footprint of EF-G within the ribosome and induces the AcPhe-tRNA binding to the P-site, but not to the A-site. Similar, but of lower degree, was the effect 5S rRNA labelling by ABA-spermine. In summary, a comprehensive view of the 5S rRNA nucleotide residues involved in spermine binding is gained by the present study. The results further suggest that binding of spermine to 5S rRNA causes conformational changes in certain regions of the molecule. These changes, although not of the extreme amplitude, enabled us to investigate the functional role of 5S rRNA. In light of this role, it seems that PTase center and EF-G binding center are able to coordinate their functions by transmission of allosteric signals through 5S rRNA.

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg2+ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca2+-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg2+, and (5) reductive alkylation of vimentin with pyridoxal-5'-phosphate and NaBH4 has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.

Chapter 8 The Modified Nucleotides in Ribosomal RNA of Man and Other Eukaryotes

Advances in molecular biology are now providing the means for solving long-standing problems in microbiology. One of the best examples is the development of a rational approach to the phylogenetic classification of microorganisms, based on comparative analysis of slowly evolving molecular components, most notably ribosomal RNAs (Woese, 1987). Molecular biologists and microbiologists have been quick to recognize how rRNA sequence variation could be used to answer major questions limiting progress in microbial ecology. Only a few years after the initial rRNA-based phylogenetic observations were published (Woese and Fox, 1977), the 16S rRNA molecule was used to characterize Prochloron, an uncultivated symbiont of marine invertebrates (Seewaldt and Stackebrandt, 1982), and the smallest ribosomal RNA molecule, 5S rRNA, was used to analyze the composition of a few simple microbial communities (Stahl et al., 1984, 1985; Lane et al., 1985b). Some further ecologic work with 5S rRNA has appeared (Colwell et al., 1989), but extensive community analysis with this molecule is complicated by the difficulty of physically separating 5S rRNAs, and by the relatively small size and thus limited information content of this molecule. In the last few years, considerable emphasis has been given in both microbial phylogeny and microbial ecology to the development of methods for studying the larger and more informative rRNAs. Most of the work has been with small ribosomal subunit rRNA (SSU rRNA, 16S in prokaryotes and 18S in eukaryotes), though a limited amount of work has been done with the larger rRNAs of large ribosomal subunits (here termed LSU rRNA, 23S in prokaryotes and 28S in eukaryotes) and with internal transcribed spacer (ITS) regions separating rRNA genes.

Multiple Functions of the 37/67-kd Laminin Receptor Make It a Suitable Target for Novel Cancer Gene Therapy

Eif6 Dosage Alleviates Activation of the Tp53 Pathway in Sbds-Deficient Cells: A Mechanism for Somatic Genetic Rescue in Shwachman-Diamond Syndrome

Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy

Lysis of Escherichia coli with a neutral detergent

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

Rapidly labeled, polydisperse RNA in rat-liver cytoplasm: Evidence that it is contained in ribonucleoprotein particles of heterogeneous size

The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated. Labelled 17-S and 26-S rRNA were prepared from cells that had been incubated with either [32P]phosphate, [Me-3H]methionine or [Me-14C]methionine. Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two-dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17-S and 26-S rRNA were performed. 17-S and 26-S rRNA contain 49 and 91 methyl groups per molecule, respectively. These values were verified in sevemral ways. The high degree of methylation of sycamore rRNA, particularly for the 26-S rRNA, contrasts with the situation in all other investigated organisms. Several methylated bases were identified. 7-Methylguanine and 5-methylcytosine both occur in 17-S and 26-S rRNA. N6-Methyladenine and N6,N6-dimethyladenine are restricted to the 17-S rRNA while 3-methyluracil and 1-methyladenine occur in the 26-S rRNA. One hypermodified uridine was also tentatively identified in the small rRNA. In 17-S rRNA, there is one copy of 7-methylguanine, N6-methyladenine and hypermodified uridine and two copies of N6,N6-dimethyladenine. 3-Methyluracil, 1-methyladenine and 5-methylcytosine occur twice, twice and three times, respectively, in 26-S rRNA. 7-Methylguanine and 5-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively. There are 40 +/- 2 and 83 +/- 3 2'-O-methylriboses per 17-S and 26-S rRNA molecule, respectively. In addition to the four 2'-O-methylnucleosides, one 2'-O-methylpseudouridine is present in the 17-S rRNA. Several lines of evidence argues for a non-random distribution of the methylriboses. In particular, one and seven Nm-Nm-Np structures occur in the 17-S and 26-S rRNA, respectively. The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and HeLa cell rRNA.

PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.

The remarkable advances in the methods for the isolation of nuclei and nucleoli of cancer cells along with the rapid evolution of techniques for purification and structural analyses of high molecular weight RNA has provided new opportunities to comparatively evaluate the constituents of tumor cells and other cells. Complete pancreatic RNase digestion was carried out on highly purified nucleolar 45S, 35S and 28S RNA and ribosomal 28S and 18S RNA of Novikoff hepatoma ascites cells which were highly labeled in vitro with 32P-orthophosphate. In addition, rat liver ribosomal 28S RNA was labeled in vivo and subjected to the same isolation and RNase digestion procedure. The resultant oligonucleotides of the digests are purine containing products with a pyrimidine 3′ terminal, altogether referred to as the pyrimidine catalog of the RNA molecule. They were separated on DEAE Sephadex A-25 at pH 7.6 according to chain length and the single components were isolated by paper electrophoresis at pH 3.5 (mono- and dinucleotides) or by rechromatography on DEAE Sephadex A-25 at pH 3.3 (hepta- to tridecanucleotides). Sequence studies were carried out by means of nucleotide analysis, complete digestion with T1 RNase and partial digestion with spleen phosphodiesterase. The oligonucleotide sequences and their frequencies were compared between the different high molecular weight RNA species of the Novikoff hepatoma and 28S ribosomal RNA of rat liver. Interestingly, marked differences were found between the substructures of nucleolar 45S, 35S and 28S RNA compared to those of ribosomal 18S RNA. The longest polypurine sequences of 28S ribosomal RNA were identical to those of 28S nucleolar RNA and no differences were found between polypurines of the Novikoff hepatoma and rat liver. However, when the frequencies were determined for the mono- and dinucleotides, significant differences appeared mainly for the dinucleotide G-Up. On the basis of a calculated chain length of 4.500 for ribosomal 28S RNA, 134 G-Up residues were calculated for the Novikoff hepatoma compared to 103 for 28S rRNA of rat liver. These results provide further evidence for defined sequence differences between high molecular weight RNA of tumor cells and other cells and possibly point the way to definitive cancer chemotherapy. Critical evaluations of potential strategies are discussed.