Evolutionary trends in 18S ribosomal RNA nucleotide sequences of rat, mouse, hamster and man.

The large T1 ribonuclease fragments of 18S ribosomal RNA from four mammalian species, rat, mouse, hamster and man, were compared by two-dimensional homochromatography fingerprinting. The nucleotide sequences of the large T1 ribonuclease fragments, polypyrimidines and polypurines which were different among the four mammalian species were determined and compared. The method used for determining nucleotide sequences utilizes 32p-labeling of oligonucleotides at their 5'-termini by polynucleotide kinase, partial digestion by ribonucleases and analysis of labeled spots by homochromatography-fingerprinting. Several examples of point mutations were detected. It was of interest that the 18S rRNA of Chinese hamster has more oligonucleotide sequences in common with those of man that rat or mouse.     

Nucleotide sequences of chloroplast 5S ribosomal ribonucleic acid in flowering plants.

Evidence for the sequence of duckweed (Lemna minor) chloroplast 5S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The possible sequence of the chloroplast 5S rRNA from three other flowering plants was deduced by complete digestion with T1 ribonuclease and comparison of the sequences of the oligonucleotide products with homologous sequences in the duckweed 5S rRNA. This analysis indicates that the chloroplast 5S rNA species differ appreciably from their cytosol counterparts but bear a strong resemblance to one another and to the 5S rRNA species of prokaryotes. Structural features apparently common to all 5S rRNA molecules are also discussed.     

The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli

Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1. A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11. The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests. It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E. coli 23 S rRNA.     

Secondary structure of Tetrahymena thermophilia 5S ribosomal RNA as revealed by enzymatic digestion and microdensitometric analysis.

The secondary structure of [32P] end-labeled 5S rRNA from Tetrahymena thermophilia (strain B) has been investigated using the enzymes S1 nuclease, cobra venom ribonuclease and T2 ribonuclease. The results, analyzed by scanning microdensitometry and illustrated by three-dimensional computer graphics, support the secondary structure model of Curtiss and Vournakis for 5S rRNA. Aberrent mobility of certain RNA fragments on sequencing gels was observed as regions of band compression. These regions are postulated to be caused by stable internal base-pairing. The molecule was probed with T2 RNase in neutral (pH 7.5) and acidic (pH 4.5) buffers and only minor structural differences were revealed. One of the helices was found to be susceptible to enzymatic attack by both the single-strand and double-strand specific enzymes. These observations are evidence for the existence of dynamic structural equilibria in 5S rRNA.     

Extensive homologies between the methylated nucleotide sequences in several vertebrate ribosomal ribonucleic acids.

The methylated nucleotide sequences in the rRNA molecules of the following vertebrate cultured cells were compared: human (HeLa); hamster (BHK/C13); mouse (L); chick-embryo fibroblast; Xenopus laevis kidney. In each species the combined 18S, 28S and 5.8S molecules possess approx. 110-115 methyl groups, and the methylated oligonucleotides released after complete digestion of the rRNA by T1 ribonuclease encompass several hundred nucleotides. "Fingerprints" of the three mammalian methyl-labelled 18S rRNA species were qualitatively indistinguishable. "Fingerprints" of digests of 28S rRNA of hamster and mouse L-cells were extremely similar to those of HeLa cells, differing in one and three methylated oligonucleotides respectively. "Fingerprints" of methyl-labelled rRNA from chick and Xenopus strongly resembled those of mammals in most respects, but differed in several oligonucleotides in both 18S and 28S rRNA. At least some of the differences between "fingerprints" appear to be due to single base changes or to the presence or absence of methyl groups at particular points in the primary sequence. The findings strongly suggest that the methylated-nucleotide sequences are at least 95% homologous between the rRNA molecules of the two most distantly related vertebrates compared, man and Xenopus laevis.     

The binding site for ribosomal protein complex L8 within 23 s ribosomal RNA of Escherichia coli

The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10. This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1. Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins. They were also subjected to nucleotide sequence analysis. The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study. Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA. It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E. coli.     

Maturation pathway for Novikoff ascites hepatoma 5.8 S ribosomal ribonucleic acid. Evidence for its presence in 32 S nuclear ribonucleic acid.

Evidence that 32 S nRNA contains 5.8 S rRNA was provided by studies on specific oligonucleotide sequences of these RNA species. Purified 32P-labeled 5.8 and 28 S rRNA and 32 S RNA were digested with T-1 ribonuclease, and the products were fractionated according to chain length by chromatography on DEAE-Sephadex A-25 at neutral pH. The oligonucleotides in Peak 8 were treated with alkaline phosphatase and the products were separated by two-dimensional electrophoresis on cellulose acetate at pH 3.5 and DEAE-paper in 7% formic acid. Seven unique oligonucleotide markers for 5.8 S rRNA including the methylated octanucleotide A-A-U-U-Gm-G-A-Gp were present in 32 S RNA but were not found in 28 S rRNA, indicating that 5.8 S rRNA is directly derived from the 32 S nucleolar precursor. These studies confirm a maturation pathway for rRNA species in which 32 S nucleolar RNA is a precursor of 5.8 S rRNA as well as 28 S rRNA.     

Conformation of methylated sequences in HeLa cell 18-S ribosomal RNA: nuclease S1 as a probe

18-S rRNA from HeLa cells was digested with nuclease S1. Under the conditions employed 15% of the total nucleotides and some 50% of the methylated nucleotides were released as low-molecular-weight products. The material which was precipitable by 70% ethanol after nuclease S1 digestion was subjected to further digestion by combined T1 plus pancreatic ribonucleases or by T1 ribonuclease alone, and fingerprints were prepared. It was found that the four sites which are modified late during ribosome maturation, and which contain base modifications, were all accessible to nuclease S1. By contrast fewer than one-half of the sites which are modified early during ribosome maturation, and which contain 2'-O-methyl groups, were accessible to nuclease S1; the remainder were protected, presumably by secondary or tertiary interactions within 18-S rRNA.     

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.     

Protein--RNA interaction in the rat liver 5S rRNA-protein L5 complex studied by digestion with ribonucleases.

Protein-RNA interactions in the 5S rRNA-protein L5 complex from rat liver ribosomes were studied by limited digestion of free and protein bound 5S rRNA with ribonuclease A and T1. In the complex with protein L5 the digestion of 5S rRNA by ribonuclease T1 is decreased at G37 and G89, whereas U38 and C39, and to a lower extent also C10 and U12 become accessible for ribonuclease A.     

Nucleotide sequences of wheat-embryo cytosol 5-S and 5.8-S ribosomal ribonucleic acids

The nucleotide sequences of wheat embryo 5.8-S and 5-S rRNAs have been determined with the use of several techniques, including classic analysis of oligonucleotides generated by ribonuclease T1 and resolution on gels of terminally labelled RNA partially degraded with ribonucleases or with chemical reagents. The sequence of wheat embryo 5.8-S rRNA was found to be (formula: see text). This sequence is compared to 5-S rRNA sequences previously published for wheat and several other angiosperms.     

16S ribosomal RNA of Escherichia coli contains a N2-methylguanosine at 27 nucleotides from the 3'' end

The 49 nucleotides fragment derived from the 3' end of 16S rRNA by cloacin DF13, is not cleaved by ribonuclease T1 at a guanosine residue tha is present at 27 nucleotides from the 3' terminus (position 115 in 16S rRNA). Analysis of the isolated nucleotide indicates that it is a modified G residue. In vivo labeling with (3H)methionine shows that this G is methylated and co-chromatography with markers reveals that it is N2-methylguanosine.     

Electron microscopic mapping of secondary structures in bacterial 16S and 23S ribosomal ribonucleic acid and 30S precursor ribosomal ribonucleic acid

Electron microscopy revealed reproducible secondary structure patterns within partially denatured 16S and 23S ribosomal ribonucleic acid (rRNA) from Escherichia coli. When prepared with 50% formamide-100 mM ammonium acetate, 16S rRNA included two small hairpins that appeared in over 50% of all molecules. Three open loops were observed with frequencies of less than 25%. In contrast, 23S rRNA included a terminal open loop and two additional large structures in over 75% of all molecules. These secondary structure patterns were conserved in the 16S and 23S rRNA from Pseudomonas aeruginosa. The secondary structure of the 30S precursor rRNA from the ribonclease III-deficient E. coli mutant AB105 was mapped after partial denaturation in 70% formamide-100 mM ammonium acetate. Two large open loops were superimposed on the 16S and 23S rRNA secondary structure patterns. These loops were the most frequent structures found on the precursor, and their stems coincided with ribonuclease III cleavage sites. A tentative 5'-3 orientation was determined for the secondary structure patterns of 16S and 23S rRNA from their relative locations within 30S precursor rRNA. The relation of secondary structure to ribosomal protein binding and ribonuclease III cleavage is discussed.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.     

Wheat embryo mitochondrial 18S ribosomal RNA: evidence for its prokaryotic nature.

We present a catalog of sequences of oligonucleotides produced by T1 ribonuclease digestion of 32P-labeled small-ribosomal-subunit RNA ("18S rRNA) isolated from purified wheat embryo mitochondria. This catalog is compared to catalogs published for prokaryotic and chloroplast 16S rRNAs and to preliminary results for wheat cytosol 18S rRNA. These comparisons indicate that: (1) wheat mitochondrial 18S rRNA is clearly prokaryotic in nature, showing significantly more sequence homology with 16S rRNAs than can be expected to arise by chance (p less than 0.000001); (2) shared oligonucleotide sequences include an especially high proportion of those identified as conserved in the evolution of prokaryotic rRNAs; and (3) wheat embryo mitochondrial and cytosol 18S rRNAs retain no more, and perhaps less, than the minimum sequence homology detectable by this sensitive method. These results argue in favor of an endosymbiotic origin for mitochondria.     

The connection between ribophagy, autophagy and ribosomal RNA decay

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.     

The presence of a high-molecular-weight (guanine-plus-cytosine)-rich segment at the 3'' end of rabbit 28S ribosomal ribonucleic acid.

The 3' hydroxyl end of 28S L-rRNA (major RNA species of the larger subribosomal particle) was labelled by coupling its 2-hydroxy-3-naphthoic acid hydrazine with diazotized [3H]aniline. The RNA was hydrolysed partially with ribonuclease T1 and fractionated on Sephadex G-200. The results show that a highly structured segment with 78% G+C content and a number-average molecular weight of at least 1.0x10(5)-1.8x10(5) is located at the 3' hydroxyl end of the 28S rRNA molecule.     

Quantitative analysis of rat liver nucleolar and nucleoplasmic ribosomal ribonucleic acids.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.