Nucleotide sequence of 5S ribosomal RNA from Lingula anatina. A study on the molecular evolution of 5S ribosomal RNA from a living fossil

By chromatography on columns of DEAE-Sephadex A-50 and Sephadex G-100, and electrophoresis on polyacrylamide gel, 5S rRNA was purified from a low-molecular-weight RNA fraction extracted from the total tissues of Lingula anatina. Complete digests of the 5S rRNA with RNase T1 [EC 3.1.4.8] and pancreatic RNase [EC 3.1.4.22] were sequenced by conventional column chromatography procedures. The nucleotide sequence of this RNA was determined mainly by a chemical method for sequencing the RNA 3' end-labeled with 32P (1), with the complement of the oligonucleotide catalog obtained by the complete RNase digestions of the RNA. By comparing the sequences of several invertebrate, vertebrate, and Chlorella 5S rRNAs, a phylogenic tree of the rRNAs was constructed and the time of divergence of Lingula was estimated.     

A proposed nucleotide sequence for the 5S ribosomal ribonucleic acid of rainbow trout (Salmo gairdneri).

Rainbow trout cell cultures have been exposed to 32P-labelled inorganic phosphate and the labelled RNA has been isolated. The 5S ribosomal ribonucleic acid (5S rRNA) was purified by polyacrylamide gel electrophoresis, then digested with RNase T1 or pancreatic RNase. The products of complete digestion were separated and their sequences determined. These analyses have allowed a sequence to be proposed which differs in eight positions from that of mammalian 5S rRNAs.     

Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Identification of a cytidine-specific ribonuclease from chicken liver

Rapid RNA sequencing technology was used to determine if the base specificities of an RNase recently purified from chicken liver would prove useful for RNA sequence analysis. Escherichia coli 5 S [5'-32P]rRNA or yeast 5.8 S [5'-32P]rRNA was digested with the enzyme and this digest, along with digests derived from RNases of known specificity (U2, T1, T2) were subjected to electrophoresis through denaturing polyacrylamide slab gels. Following autoradiography, the banding patterns arising from the activity of each enzyme were compared, and the base specificity of the unknown RNase was established. The chicken liver RNase was found to have a marked preference for phosphodiester bonds containing cytidylic acid residues, a property which should make the enzyme useful for distinguishing between pyrimidines in RNA sequencing.     

The 3''-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3''-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.     

Nuclear RNase MRP is required for correct processing of pre-5.8S rRNA in Saccharomyces cerevisiae.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype.Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.     

Double-stranded RNA specific nuclease from germinating embryos ofPennisetum typhoides

A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl milletPennisetum typhoides. The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography. The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide [³H]poly(rA) · poly(rU); the degradation of [³H]poly(rC) was fourteen fold lower under the same assay conditions. Further more, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs. Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide. Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity. In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58%. Heating the enzyme for 20 min at 50°C resulted in drastic loss of both enzyme activities. The ds RNase showed maximum activity in the pH range of 6.5 to 7.5. The enzyme actsin vitro onE. coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs.     

The use of RNAse H for digestion of alkylated 16S rRNA at the binding sites of addressed reagents--oligodeoxyribonucleotide derivatives

It is demonstrated that 16S rRNA, complementary-addressed labelled with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligonucleotides d(pACCTTGTT)rA and d(pTTTGCTCCCC)rA, can be cleaved by RNase H within the adducts, resulted from the modification. Comparative study of the 16S rRNA cleavage with RNase H within the above--mentioned covalent adducts, on the one hand, and within heteroduplexes with the same oligodeoxyribonucleotides, on the other, showed that(i) the complementary-addressed modification proceeds both in perfect and non-per ect complexes; (ii) 16S rRNA is cleaved by RNase H within both perfect and non-perfect complexes resulted from the alkylation, non-perfect complexes being considerably stabilized by the covalent bond between the reagent and the RNA; (iii) non-perfect complexes of 16S rRNA with the free oligodeoxyribonucleotides are unstable even at the high oligonucleotide concentration, so that no cleavage of 16S rRNA in such duplexes is observed. The approach based on cleavage of RNA within covalent adducts resulted from the complementary-addressed RNA modification may be used for fragmentation of RNA molecule in the addressed reagent's binding site.     

Nucleotide sequence of 5S ribosomal RNA from Aspergillus nidulans and Neurospora crassa

The nucleotide sequences of 5S rRNA molecules isolated from the cytosol and the mitochondria of the ascomycetes A. nidulans and N. crassa were determined by partial chemical cleavage of 3'-terminally labelled RNA. The sequence identity of the cytosolic and mitochondrial RNA preparations confirms the absence of mitochondrion-specific 5S rRNA in these fungi. The sequences of the two organisms differ in 35 positions, and each sequence differs from yeast 5S rRNA in 44 positions. Both molecules contain the sequence GCUC in place of GAAC or GAUY found in all other 5S rRNAs, indicating that this region is not universally involved in base-pairing to the invariant GTpsiC sequence of tRNAs.     

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.