In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA.

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'-terminal sequence of the VSV New Jersey genome RNA was detemined and found to contain the sequence- Py-G-UOH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA''s of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

Complete nucleotide sequence of tobacco streak virus RNA 3

Double-stranded cDNA of in vitro polyadenylated tobacco streak virus (TSV) RNA 3 has been cloned and sequenced. The complete primary structure of 2,205 nucleotides reveals two open reading frames flanked by a leader sequence of 210 bases, an intercistronic region of 123 nucleotides and a 3'-extracistronic sequence of 288 nucleotides. The 5'-terminal open reading frame codes for a Mr 31,742 protein, which probably corresponds to the only in vitro translation product of TSV RNA 3. The 3'-terminal coding region predicts a Mr 26,346 protein, probably the viral coat protein, which is the translation product of the subgenomic messenger, RNA 4. Although the coat proteins of alfalfa mosaic virus (A1MV) and TSV are functionally equivalent in activating their own and each others genomes, no homology between the primary structures of those two proteins is detectable.     

Leader sequence distinguishes between translatable and encapsidated measles virus RNAs.

The 3'-terminal 55 nucleotides of the negative-strand measles virus RNA genome called the leader sequence is not transcribed into a detectable distinct RNA product. Most of the monocistronic N and bicistronic N-P RNAs lack the leader sequence. However, a subpopulation of the N and N-P RNAs and all of the antigenomes possess this leader. Here, we show that leader-containing subgenomic RNAs are functionally distinct from their leaderless counterparts. In measles virus-infected cells, leaderless monocistronic N and bicistronic N-P RNAs were associated with polysomes. By contrast, leader-containing N and N-P RNAs were found exclusively in nonpolysomal ribonucleoprotein complexes that were resistant to RNase and had a buoyant density of 1.30 g/ml, the same as that of antigenomic ribonucleoprotein complexes. Both antigenomic and subgenomic ribonucleoprotein complexes were specifically immunoprecipitated by antiserum against the N protein, and leaderless RNAs were not found in these complexes. These findings suggest that measles virus distinguishes RNAs destined for encapsidation or translation by the presence or absence of a leader sequence.     

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.     

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.     

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5'-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3'-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5'- and 3'-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.     

Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA.

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.     

Representation of the leader region of Rous sarcoma virus genome RNA in double-stranded RNA produced in virus-transformed cells

Restriction fragments of recombinant plasmids containing a proviral sequence of Rous sarcoma virus (RSV) were Southern hybridized with double-stranded (ds) RNA isolated from the cells transformed with RSV. Hybridization data show that the major subpopulation of dsRNA molecules is homologous to the 5'-end region of the viral genome including the leader sequence. We have analysed the RNAs of RSV-transformed cells by the Northern procedure hybridizing them with the proviral fragment containing double long terminal repeats. The results demonstrate that the 14-16S RNA fraction is enriched in sequences which are homologous to the proviral end regions. We consider this RNA fraction to be homologous to the 5'-terminal region of the viral genome and (or) to its antisense strand.     

Analysis of the genome structure of tobacco rattle virus strain PSG.

The sequence of the 3'-terminal 2077 nucleotides of genomic RNA 1 and the complete sequence of genomic RNA 2 of tobacco rattle virus (TRV, strain PSG) has been deduced. RNA 2 (1905 nucleotides) contains a single open reading frame for the viral coat protein (209 amino acids), flanked by 5'- and 3'-noncoding regions of 570 and 708 nucleotides, respectively. A subgenomic RNA (RNA 4) was found to lack the 5'-terminal 474 nucleotides of RNA 2 and is the putative messenger for coat protein. The deduced RNA 1 sequence contains the 3'-terminal part of a reading frame that probably corresponds to the TRV 170K protein and reading frames for a 29K protein and a 16K protein. Proteins encoded by the first two reading frames show significant amino acid sequence homology with corresponding proteins encoded by tobacco mosaic virus. Subgenomic RNAs 3 (1.6 kb) and 5 (0.7 kb) were identified as the putative messengers for the 29K and 16K proteins, respectively. At their 3'-termini all PSG-RNAs have an identical sequence of 497 nucleotides; at the 5'-termini homology is limited to 5 to 10 bases.     

A candidate U1 small nuclear RNA for trypanosomatid protozoa.

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.     

Probable involvement of 3'-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A possibility of involvement of 3'-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 is accessible for complementary interactions in 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA when added to wheat germ cell-free protein synthesizing system were found to specifically inhibit translation of uncapped reporter mRNA coding for beta-glucuronidase, which bears in the 5'-untranslated region (UTR) a leader sequence of potato virus Y (PVY) genomic RNA possessing fragments complementary to the region 1777-1811. It was shown that a sequence corresponding to nucleotides 291-316 of PVY, which is complementary to a major portion of the 3-terminal 18S rRNA segment 1777-1808, when placed into 5'-UTR, is able to enhance translational efficiency of the reporter mRNAs. The results obtained suggest that complementary interactions between mRNA 5'-UTR and 18S rRNA 3'-terminal segment can take place in the course of cap-independent translation initiation.     

Structure of the 5'-terminal untranslated region of the genomic RNAs from two strains of alfalfa mosaic virus.

We report the sequences of the 5'-terminal regions of the 3 Alfalfa Mosaic Virus genomic RNAs for the Strasbourg strain (AlMV-S) and for a new isolate, AlMV-B; they are compared to similar data obtained by Koper-Zwarthoff et al. (Nucleic Acids Res. 1980, 8, 5635-5647) for strain 425. The structure of these leaders is highly conserved in RNAs 1 and 2. The length of the leader is 102, 100 and 101 nucleotides in RNA1 for strains S, B and 425 respectively; 55 and 56 in RNA2 for strains S and B respectively. In RNA3 however, there are important differences near the 5'-terminus between strain S and the other two: The total leader length is 258 nucleotides for strain S and 242 for strain B. The secondary structure models show a conserved hairpin near the 5'-end of each genomic RNA of AlMV-S. This hairpin is inexistent in RNA3 of the B and 425 strains. The degree of base-pairing increases with leader length. The initiator codon is located in a single stranded region in RNA2 whereas it is found in a hairpin stem in RNA 3.