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.     

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 conformation of chicken, rat and human U1A RNAs in solution.

Chicken, rat and human U1A RNAs in solution, were examined for secondary structure, using several methods including hydrolysis by various nucleases, hybridization to DNA oligomers and analysis of fragment interactions. The experimental results showed that the three U1A RNAs have the same structure, stable over a wide range of pH and ionic conditions. They allowed the selection of one out of several possible models constructed from the data of primary structure. This model is characterized by 4 hairpins and two single-stranded regions, the two hairpins from the 3' part of the molecule bearing very stable stems. In addition, the experimental results showed that in contrast to the 5' half of the molecule, the 3' half has a compact conformation probably stabilized by tertiary interactions. The 5' end of U1A RNA is accessible and free of base-pairing so that it might base-pair with regions of other RNA molecules, for instance, with the extremities of introns as has been recently proposed in a model of splicing.     

Plant small nuclear RNAs. V. U4 RNA is present in broad bean plants in the form of sequence variants and is base-paired with U6 RNA.

U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles. It occurs in broad bean in the form of a number of sequence variants. Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long. Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae. Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half. The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far. Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules. A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA. This is the first report on the structure of a plant U4 RNA.     

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.     

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.     

Four novel U RNAs are encoded by a herpesvirus

Marmoset T lymphocytes transformed by herpesvirus saimiri contain the first virally encoded U RNAs (called HSURs) to be identified. HSURs assemble into small nuclear ribonucleoproteins of low abundance (less than or equal to 2 x 10(4) copies/cell). They bind proteins with Sm determinants and acquire a 5' trimethylguanosine cap structure. The sequences of HSUR 1 (143 nucleotides), HSUR 2 (115 nucleotides), HSUR 3 (76 nucleotides), and HSUR 4 (106 nucleotides) are related to each other but are distinct from any previously characterized cellular U RNA. The viral genes encoding the HSURs possess conserved enhancer, promoter, and 3' end formation signals unique to U RNA genes. HSUR 1 and HSUR 2 have a similar 5' end sequence that exhibits perfect complementarity to the highly conserved AAUAAA polyadenylation signal. Oligonucleotide directed RNAase H degradation indicates that this 5' end region is available for base pairing interactions within the HSUR 1 and HSUR 2 snRNP particles.     

Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs

Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.     

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.     

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.     

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.     

Nucleotide sequence of nucleolar U3B RNA.

U3A, U3B, and U3C are three distinct molecular weight nucleolar RNAs present in Novikoff hepatoma ascites cells. The primary nucleotide sequence of U3B, the most prominent of these U3 species, was determined. Purified U3B RNA was subjected to various enzymatic digestion procedures, including digests of 32P-labeled U3B RNA, RNA ligase, and polynucleotide kinase labeling, for determination of its primary sequence which is: (formula: see text). The 5'-terminus of the RNA has a "cap" and localized purine-rich regions were found near the 3'-terminus, which have been incorporated into a hydrogen-bonded region in a proposed secondary structure of the molecule.     

S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA

We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.     

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.     

The small nuclear RNAs of Drosophila

We have investigated the sequences of the major small nuclear RNAs of Drosophila cultured cells, with the objective of elucidating phylogenetically conserved primary and secondary structures by comparison of the data with previously determined sequences of these RNAs in vertebrate species. Our results reveal striking degrees of conservation between each Drosophila RNA and its vertebrate cognate, and also demonstrate blocks of homology among the Drosophila small nuclear RNAs, as previously described for vertebrates. The most conserved features include the 5' terminal region of U1 RNA, though to function in pre-mRNA splicing, most of the regions of U4 RNA recently implicated in 3' processing of pre-mRNA, and the major snRNP protein binding site ("domain A") that is also shared by vertebrate U1, U2, U4 and U5 RNAs. Several other conserved features have been revealed, suggesting additional regions of functional significance in these RNAs and also providing further insights into the evolutionary history of the small nuclear RNAs.     

Effects of mutations of the initiation nucleotides on hepatitis C virus RNA replication in the cell

Replication of nearly all RNA viruses depends on a virus-encoded RNA-dependent RNA polymerase (RdRp). Our earlier work found that purified recombinant hepatitis C virus (HCV) RdRp (NS5B) was able to initiate RNA synthesis de novo by using purine (A and G) but not pyrimidine (C and U) nucleotides (G. Luo et al., J. Virol. 74:851-863, 2000). For most human RNA viruses, the initiation nucleotides of both positive- and negative-strand RNAs were found to be either an adenylate (A) or guanylate (G). To determine the nucleotide used for initiation and control of HCV RNA replication, a genetic mutagenesis analysis of the nucleotides at the very 5' and 3' ends of HCV RNAs was performed by using a cell-based HCV replicon replication system. Either a G or an A at the 5' end of HCV genomic RNA was able to efficiently induce cell colony formation, whereas a nucleotide C at the 5' end dramatically reduced the efficiency of cell colony formation. Likewise, the 3'-end nucleotide U-to-C mutation did not significantly affect the efficiency of cell colony formation. In contrast, a U-to-G mutation at the 3' end caused a remarkable decrease in cell colony formation, and a U-to-A mutation resulted in a complete abolition of cell colony formation. Sequence analysis of the HCV replicon RNAs recovered from G418-resistant Huh7 cells revealed several interesting findings. First, the 5'-end nucleotide G of the replicon RNA was changed to an A upon multiple rounds of replication. Second, the nucleotide A at the 5' end was stably maintained among all replicon RNAs isolated from Huh7 cells transfected with an RNA with a 5'-end A. Third, initiation of HCV RNA replication with a CTP resulted in a >10-fold reduction in the levels of HCV RNAs, suggesting that initiation of RNA replication with CTP was very inefficient. Fourth, the 3'-end nucleotide U-to-C and -G mutations were all reverted back to a wild-type nucleotide U. In addition, extra U and UU residues were identified at the 3' ends of revertants recovered from Huh7 cells transfected with an RNA with a nucleotide G at the 3' end. We also determined the 5'-end nucleotide of positive-strand RNA of some clinical HCV isolates. Either G or A was identified at the 5' end of HCV RNA genome depending on the specific HCV isolate. Collectively, these findings demonstrate that replication of positive-strand HCV RNA was preferentially initiated with purine nucleotides (ATP and GTP), whereas the negative-strand HCV RNA replication is invariably initiated with an ATP.