Homologies in eukaryotic 5.8S ribosomal RNA.

The primary nucleotide sequence of Novikoff hepatoma ascites cell 5.8S rRNA (also known as 5.5 or 7S RNA) has been determined to be:

This sequence is 75% homologous with the primary nucleotide sequence of yeast 5.8S rRNA and 100% homologous with oligonucleotide marker fragments from HeLa cell RNA. In constrast, only limited homology is evident with oligonucleotides from 5.8S RNA of several flowering plants and many of the characteristic fragments differ.     

Inhibition of protein synthesis by anti-5.8 S rRNA oligodeoxyribonucleotides

To examine the role of the 5.8 S rRNA in ribosome function, oligodeoxyribonucleotides, complementary to chemically accessible sequences, were incubated with rabbit reticulocyte or wheat germ extracts undergoing protein synthesis in vitro. Significant and reproducible inhibitions were observed with several different oligonucleotides, the most inhibitory being specific for the universally conserved GAAC sequence. Mutant or heterologous sequences were substantially less inhibitory, results which clearly implicate the 5.8 S rRNA in the inhibitory process and are consistent with the possibility that the 5.8 S rRNA plays an important role in the binding of tRNA.     

Sequence homologies in mammalian 5.8S ribosomal RNA.

Oligonucleotide products of complete pancreatic or T1 RNase digestion or partial T1 RNase digestion of HeLa cell (human) and MPC-11 cell (mouse) 5.8S rRNA are identical with those obtained from Novikoff hepatoma (rat) 5.8S rRNA except for minor differences at the termini. pCp is the only major 5' terminus of both human and mouse RNAs; both pGp and pCp 5' termini were found in rat 5.8S RNA. Furthermore, HeLa cells contain C-U-U at the 3' end rather than the C-U terminus of mouse and rat. The results indicate that the nucleotide sequence has been highly conserved during the evolution of mammals and suggest that, as reported for 5S rRNA, this sequence is essentially constant throughout the Mammalia.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.     

Binding of rat ribosomal proteins to yeast 5.8S ribosomal ribonucleic acid.

5.8 S RNA-protein complexes were prepared using purified yeast 5.8 S RNA and proteins from the large ribosomal subunit of rat liver. Formation of such hybrid complexes, as measured by Millipore filtration, was dependent on protein concentration. Binding of proteins to the RNA could approach saturation. Such complexes were isolated from sucrose density gradient centrifugation and shown to contain proteins L6, L8, L19, L35 and L35a. These proteins were identified by their molecular weights on polyacrylamide gels containing dodecylsulfate and their mobilities on two dimensional polyacrylamide gels.     

Transcriptional organization of the 5.8S ribosomal RNA cistron in Xenopus laevis ribosomal DNA.

Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.8S rRNA cistron lies within the transcribed region that links the 18S and 28S rRNA cistrons.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

Higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA synthesized in vitro

Dimethylsulfate, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene-sulfonate, RNase T1 and RNase V1 have been used as structure-sensitive probes to examine the higher-order structure of the 5.8 S rRNA sequence within the yeast 35 S precursor ribosomal RNA molecule. Data produced have been used to evaluate several theoretical structure models for the 5.8 S rRNA sequence within the precursor rRNA. These models are generated by minimum free energy calculations. A model is proposed that accommodates 83% of the residues experimentally shown to be in either base-paired or single-stranded structure in the correct configuration. Several alternative suboptimal secondary structures have been evaluated. Moreover, the chemical reactivities of several residues within the 5.8 S rRNA sequence in the precursor rRNA molecule differ from those of the corresponding residues in the mature rRNA molecule. This finding provides experimental evidence to support the notion that the 5.8 S rRNA sequence within the precursor rRNA undergoes structural reorganization following rRNA processing.     

Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista.     

Equilibrium and kinetics of thermal unfolding of yeast 5.8S ribosomal RNA in aqueous salt solutions

Equilibrium and kinetics of thermal melting of yeast 5.8S ribosomal RNA in aqueous NaCl were investigated by differential thermal melting and temperature jump methods. Two peaks were observed in each of the melting curves at 1 mM-1 M Na⁺ and linearity between each melting temperature T_m and log[Na⁺] was found at [Na⁺> 10 mM. From the difference spectrum ratio, $\text{d}\text{A}{}_{280}\text{d}\text{A}{}_{260}$, the G-C content in the local structures was calculated to be 91 and 56%. The temperature jump to 70–85°C in aqueous 30 mM Na⁺ of the RNA solution induced first-order kinetics, from which the kinetically determined melting curve was calculated. The curve could be approximately described in a Gaussian form with a T_m which agrees well with the high T_m in the static melting curve at 30 mM Na⁺. The kinetic properties of the reaction indicated a double helix-coil transition. However, the temperature jump to 20–60°C did not induce monophasic kinetics. The kinetic amplitude of the slow component showed a T_m which corresponded to the low T_m in the static melting curve at 30 mM Na⁺. The slow relaxation had the characteristics of a double helix-to-coil transition. However, contributions from very fast processes including single strand unstacking, were most noticeable in the low temperature melting region of the static curve. The thermodynamic parameters of both transitions from double helix to coil were analysed in detail. Both activation energies for helix formation were negative, and the nucleation is thought to follow a process similar to that in oligonucleotides. Values of T_m and enthalpy change of both helix-coil transitions indicated the cloverleaf model as the most plausible one for some limited regions of yeast 5.8S RNA among the previously proposed models: burp gun, cloverleaf and Rubin's models.     

The sequence of the 5.8 S ribosomal RNA of the crustacean Artemia salina. With a proposal for a general secondary structure model for 5.8 S ribosomal RNA.

We report the primary structure of 5.8 S rRNA from the crustacean Artemia salina. The preparation shows length heterogeneity at the 5'-terminus, but consists of uninterrupted RNA chains, in contrast to some insect 5.8 S rRNAs, which consist of two chains of unequal length separated in the gene by a short spacer. The sequence was aligned with those of 11 other 5.8 S rRNAs and a general secondary structure model derived. It has four helical regions in common with the model of Nazar et al. (J. Biol. Chem. 250, 8591-8597 (1975)), but for a fifth helix a different base pairing scheme was found preferable, and the terminal sequences are presumed to bind to 28 S rRNA instead of binding to each other. In the case of yeast, where both the 5.8 S and 26 S rRNA sequences are known, the existence of five helices in 5.8 S rRNA is shown to be compatible with a 5.8 S - 26 S rRNA interaction model.