High evolutionary conservation of the secondary structure and of certain nucleotide sequences of U5 RNA.

The nucleotide sequence of chicken, pheasant, duck and Tetrahymena pyriformis U5 RNAs as well as that of new mammalian variant U5 RNAs was determined and compared to that of rat and HeLa cells U5 RNAs. Primary structure conservation is about 95% between rat and human cells, 82% between mammals and birds and 57% between the Protozoan and mammals. The same model of secondary structure, a free single-stranded region flanked by two hairpins can be constructed from all RNAs and is identical to the model previously proposed for mammalian U5 RNA on an experimental basis (1). Thus, this model is confirmed and is likely to be that of an ancestor U5 RNA. The 3' region of the U5 RNA molecule constitutes domain A, and is common to U1, U2, U4 and U5 RNAs (2). The characteristic nucleotide sequences of domain A are highly conserved throughout the phylogenetic evolution of U5 RNA suggesting that they are important elements in the function of the four small RNAs. Another region of high evolutionary conservation is the top part of the 5' side hairpin whose conserved sequence is specific to U5 RNA. It might participate in the particular function of U5 RNA.     

The nuclear 5S RNAs from chicken, rat and man. U5 RNAs are encoded by multiple genes

Preparations of chicken, rat and human nuclear 5S RNA contain two sets of molecules. The set with the lowest electrophoretic mobility (5Sa) contains RNAs identical or closely related to ribosomal 5S RNA from the corresponding animal species. In HeLa cells and rat brain, we only detected an RNA identical to the ribosomal 5S RNA. In hen brain and liver, we found other species differing by a limited number of substitutions. The results suggest that mutated 5S genes may be expressed differently according to the cell type. The set with the highest mobility corresponds to U5 RNA. In both rat brain and HeLa cells, U5 RNA was found to be composed of 4 and 5 different molecules respectively (U5A, U5B1-4) differing by a small number of substitutions or insertions. In hen brain, no U5B was detected but U5A' differing from U5A by the absence of the 3'-terminal adenosine. All the U5 RNAs contain the same set of modified nucleotides. They also have the same secondary structure which consists of two hairpins joined together by a 17 nucleotide long single-stranded region. The 3' half of the molecule has a compact conformation. Together, the results suggest that U5 RNAs are transcribed from a multigene family and that mutated genes may be expressed as far as secondary structure is conserved. The conformation of U5 RNA is likely to be related to its function and it is of interest to mention that several similarities of structure are found between U5 and U1A RNA.     

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

We previously reported common structural features within the 3'-terminal regions of U1, U4, and U5 RNAs. To check whether these features also exist in U2 RNA, the primary and secondary structures of the 3'-terminal regions of chicken, pheasant, and rat U2 RNAs were examined. Whereas no difference was observed between pheasant and chicken, the chicken and rat sequences were only 82.5% homologous. Such divergence allowed us to propose a unique model of secondary structure based on maximum base-pairing and secondary structure conservation. The same model was obtained from the results of limited digestion of U2 RNA with various nucleases. Comparison of this structure with those of U1, U4, and U5 RNAs shows that the four RNAs share a common structure designated as domain A, and consisting of a free single-stranded region with the sequence Pu-A-(U)n-G-Pup flanked by two hairpins. The hairpin on the 3' side is very stable and has the sequence Py-N-Py-Gp in the loop. The presence of this common domain is discussed in connection with relationships among U RNAs and common protein binding sites.     

The yeast homologue of U3 snRNA.

snR17, one of the most abundant capped small nuclear RNAs of Saccharomyces cerevisiae, is equivalent to U3 snRNA of other eukaryotes. It is 328 nucleotides in length, 1.5 times as long as other U3 RNAs, but shares significant homology both in nucleotide sequence and in predicted secondary structure. Human scleroderma antiserum specific to nucleolar U3 RNP can enrich snR17 from sonicated yeast nuclear extracts. Unlike other yeast snRNAs which are encoded by single copy genes, snR17 is encoded by two genetically unlinked genes: SNR17A and SNR17B. The RNA snR17A is more abundant than snR17B. Deleting one or other of the genes has no obvious phenotypic effect, except that the steady-state level of snR17B is increased in snr17a- strains. Haploid strains with both genes deleted are inviable, therefore yeast U3 is essential.     

Plant small nuclear RNAs. Nucleolar U3 snRNA is present in plants: partial characterization

Nuclei, isolated from a number of plant species by either of two independent, newly developed methods, regularly contained a common set of low-molecular-mass RNAs. Partial characterization of these RNAs, based on cell fractionation, polyacrylamide gel electrophoretic and chemical sequencing techniques, as well as comparison with literature data, revealed that, in addition to tRNA, 5S RNA and 5.8S RNA, plant nuclei contain two families of low-molecular-mass RNAs, that are counterparts of vertebrate U1 and U5 RNAs respectively, and three individual low-molecular-mass RNA species. One of these may be related to vertebrate U6 RNA. The two others are true eukaryotic U2 and U3 RNAs, respectively, on the basis of the following lines of evidence obtained from analyses of broad bean nuclear RNAs. The 3'-end portion (121 nucleotides sequenced) of broad bean U2 RNA shows a nearly perfect sequence homology with that of authentic pea U2 RNA. Broad bean U3 RNA is localized in the nucleolus and its 3'-end portion (164 nucleotides sequenced) (a) shows sequence homology with that of both rat U3 RNA (48%) and Dictyostelium D2 RNA (39%), (b) has a secondary structure which fits perfectly that proposed for both rat U3 RNA and Dictyostelium D2 RNA, and (c) contains the specific sequence which, in a model based on the primary structure of rat U3 RNA, is supposed to be involved in the processing of eukaryotic 32S pre-ribosomal RNA. This is the first report on the occurrence in plants of nucleolar U3 RNA.     

The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes.

We have isolated the gene coding for the U2 analogue in trypanosomes. The 148 nucleotide long U2 RNA is capped and transcribed from a single copy gene. The 5' half of the molecule is highly homologous to mammalian U2 RNA, while the 3' half does not show any significant sequence homology with the mammalian counterpart. Nevertheless, the trypanosome U2 RNA can be folded into a secondary structure resembling the one proposed for U2 RNA. The presence of a U2 analogue and most likely other U RNAs in trypanosomes suggests that splicing is involved at some point in the maturation of mRNA. Possible interactions of the U2 RNA with the spliced leader RNA are considered.     

Two 5S genes are expressed in chicken somatic cells.

Two 5S RNA species were detected in chicken cells. 5S I RNA has the nucleotide sequence of chicken 5S RNA previously published by Brownlee et al. (1) and 5S II RNA differs from it by 10 mutations. The secondary structure of both species is compatible with that proposed for other eukaryotic 5S RNAs. 5S II RNA represents 50-60% of 5S I RNA. Both species were found in total chicken liver and brain and were present in polysomes in the same relative proportions. Only one 5S RNA species could be detected in rat liver and HeLa cells. Chicken is the first vertebrate described so far in which two 5S RNA genes are expressed in somatic cells.     

Nucleotide sequences of nuclear U1A RNAs from chicken, rat and man.

The methods of enzymatic and chemical treatment of end-labeled RNA were applied to the determination of the nucleotide sequence of chicken and man U1A RNA and to the reexamination of that of rat U1A RNA. The chemical method allowed the easy demonstration of the cap structure. All three RNA were 165 nucleotide long. Two hitherto non described modified pyrimidines were detected close to the 5' end. Only 9 base substitutions were observed from chicken to man indicating high degree of conservation of U1A RNA through evolution.     

Extended secondary structure in 5S rRNAs from a sulphur metabolizing archaebacterium, Thermococcus celer

While this sequence shares a significant homology with the 5S RNAs of other archaebacteria and is consistent with current models for the secondary structure of 5S RNAs, it contains three unusual features. The G + C content (72-74%) is significantly higher than other 5S RNAs; the secondary structure is distinguished by unusually stable and extended helical structures and, most important, there is evidence for sequence heterogeneity in the form of complementary base substitutions and precursor processing. This supports recent evidence (Newmann, H., Gierl, A., Tu, J., Leibrock, J., Staiger, D. and Zillig, W. (1983) Mol. Gen. Genet. 192, 66-72) that, like many of the higher eukaryotes, this group of sulphur-metabolizing bacteria may contain multiple 5S RNA genes.     

U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs

I have determined the structure of the gene from Saccharomyces cerevisiae coding for the yeast homolog of vertebrate U2 snRNA. Surprisingly, the RNA is 1175 nucleotides long, six times larger than U2 RNAs from other organisms, including Schizosaccharomyces pombe. Nearly 100 nucleotides of the large RNA share sequence homology and potential secondary structure with metazoan U2. The large RNA also contains homology to vertebrate U4, U5, and U6 snRNAs, implying a "poly-snRNP" structure for the RNP containing the large RNA. The gene LSR1, encoding the large RNA, is essential for growth, suggesting that the yeast spliceosome can be dissected using genetic approaches. The different organization of spliceosomal RNA may underlie differences in splicing between yeast and metazoans.     

Widespread RNA 3'-end oligouridylation in mammals

Nontemplated 3'-end oligouridylation of RNA occurs in many species, including humans. Unlike the familiar phenomenon of polyadenylation, nontemplated addition of uridines to RNA is poorly characterized in higher eukaryotes. Recent studies have reported nontemplated 3'-end oligouridylation of small RNAs and mRNAs. Oligouridylation is involved in many aspects of microRNA biology from biogenesis to turnover of the mature species, and it may also mark long mRNAs for degradation by promoting decapping of the protective 5'-cap structure. To determine the prevalence of oligouridylation in higher eukaryotes, we used next-generation sequencing technology to deeply examine the population of small RNAs in human cells. Our data revealed widespread nontemplated nucleotide addition to the 3' ends of many classes of RNA, with short stretches of uridine being the most frequently added nucleotide.     

Isolation from rat liver and sequence of a RNA fragment containing 32 nucleotides from position 5 to 36 from the 3'' end of ribosomal 18S RNA.

Crude tRNA isolated from rat liver by the method of Rogg et al. (Biochem. Biophys. Acta 195, 13-15 1969) contains N6-dimethyladenosine (m6-2A) and was therefore fractionated in order to identify the m6-2A-containing RNAs. A unique species of RNA was purified which contained all the m62A present in the crude tRNA. Sequence analysis by postlabeling with gamma-32p-ATP and polynucleotide kinase revealed that this RNA represents the 32 nucleotides AAGGUUUC(C)U GUAGGUGm62Am62ACCUGCGGAAGGAUC from position 5 to 36 of the 3' terminus of ribosomal 18S RNA. The 36 nucleotide long sequence from the 3' end of rat liver 18S rRNA exhibits extensive homology with the corresponding sequence of E. coli 16S rRNA and with the 21 nucleotide long 3' terminal sequence so far known from Saccharomyces carlsbergensis 17S rRNA. A heterogeneity in this sequence provides the first evidence on the molecular level for the existence of (at least) two sets of redundant ribosomal 18S RNA genes in the rat.     

The sequence of U3 from Schizosaccharomyces pombe suggests structural divergence of this snRNA between metazoans and unicellular eukaryotes.

We have cloned and sequenced one of the two genes encoding a 255 nucleotide small nuclear RNA from the fission yeast Schizosaccharomyces pombe. Based on the presence of four regions of primary sequence conservation and a predicted secondary structure similar to that previously proposed for human U3, we conclude that this molecule is the fission yeast homologue of this mammalian snRNA. The 5' one-third of fission yeast U3 is, however, unable to form a single stable hairpin as proposed for this region of the human RNA, but rather folds into two stem-loop structures. By analogy to fission yeast U3, we propose revised secondary structures containing two hairpins for this portion of the U3-like snRNAs from Saccharomyces cerevisiae and Dictyostelium discoideum. Thus, our data suggest that the structure of U3 snRNA has diverged in lower and higher eukaryotes.     

Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells.

The nucleotide sequence of a particular T1 oligonucleotide found in 41S and 28S RNAs of several cellular cell lines (human, mouse, rat and chicken fibroblast) but absent in 45S ribosomal RNA has been deduced. Its primary structure : A-U-U*-G*-psi-U-C-A-C-C-C-A-C-U-A-A-U-A-Gp shows the presence of a modified G residue which explains the existence of this oligonucleotide in the T1 fingerprint of 41S RNA and 28S. Its absence on the 45S RNA T1 fingerprint is accounted for by a late modification.     

An evaluation of the phylogenetic position of the dinoflagellateCrypthecodinium cohnii based on 5S rRNA characterization

Summary Partial nucleotide sequences for the 5S and 5.8S rRNAs from the dinoflagellateCrypthecodinium cohnii have been determined, using a rapid chemical sequencing method, for the purpose of studying dinoflagellate phylogeny. The 5S RNA sequence shows the most homology (75%) with the 5S sequences of higher animals and the least homology (< 60%) with prokaryotic sequences. In addition, it lacks certain residues which are highly conserved in prokaryotic molecules but are generally missing in eukaryotes. These findings suggest a distant relationship between dinoflagellates and the prokaryotes. Using two different sequence alignments and several different methods for selecting an optimum phylogenetic tree for a collection of 5S sequences including higher plants and animals, fungi, and bacteria in addition to theC. cohnii sequence, the dinoflagellate lineage was joined to the tree at the point of the plant-animal divergence, well above the branching point of the fungi. This result is of interest because it implies that the well-documented absence in dinoflagellates of histones and the typical nucleosomal subunit structure of eukaryotic chromatin is the result of secondary loss. and not anindication of an extremely primitive state, as was previously suggested. Computer simulations of 5S RNA evolution have been carried out in order to demonstrate that the above-mentioned phylogenetic placement is not likely to be the result of random sequence convergence.We have also constructed a phylogeny for 5.8S RNA sequences in which plants, animals, fungi and the dinoflagellates are again represented. While the order of branching on this tree is the same as in the 5S tree for the organisms represented, because it lacks prokaryotes, the 5.8S tree cannot be considered a strong independent confirmation of the 5S result. Moreover, 5.8S RNA appears to have experienced very different rates of evolution in different lineages indicating that it may not be the best indicator of evolutionary relationships.We have also considered the existing biological data regarding dinoflagellate evolution in relation to our molecular phylogenetic evidence.     

The nucleotide sequence of phenylalanine tRNA and 5S RNA from Rhodospirillum rubrum

The complete nucleotide sequence of tRNAPhe and 5S RNA from the photosynthetic bacterium Rhodospirillum rubrum has been elucidated. A combination of in vitro and in vivo labelling techniques was used. The tRNAPhe sequence is 76 nucleotides long, 7 of which are modified. The primary structure is typically prokaryotic and is most similar to the tRNAPhe of Escherichia coli and Anacystis nidulans (14 differences of 76 positions). The 5S ribosomal RNA sequence is 120 nucleotides long and again typical of other prokaryotic 5S RNAs. The invariable GAAC sequence is found starting at position 45. When aligned with other prokaryotic 5S RNA sequences, a surprising amount of nucleotide substitution is noted in the prokaryotic loop region of the R. rubrum 5S RNA. However, nucleotide complementarity is maintained reinforcing the hypothesis that this loop is an important aspect of prokaryotic 5S RNA secondary structure. The 5S and tRNAPhe are the first complete RNA sequences available from the photosynthetic bacteria.