Structure mapping of 5'-<sup>32</sup>P-labeled RNA with S1 nuclease

Wurst, Regina M.; Vournakis, John N.; Maxam, Allan M.

doi:10.1021/bi00614a021

Cited by 112 publications

(73 citation statements)

References 43 publications

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“…Analysis of secondary structural features by the use of the single-strand-specific Si nuclease (22,28,41) supports the presence of the predicted stem and loop structures. Partial cleavage of the positive strand of LA dsRNA with SI nuclease resulted in sensitivity at positions 1 to 6, 11 to 14, and 20 to 28 (Fig.…”

mentioning

confidence: 65%

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Thiele¹,

Hannig²,

Leibowitz³

1984

Mol. Cell. Biol.

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The L double-stranded (ds) RNA component of Saccharomyces cerevisiae may contain up to three dsRNA species, each with a distinct sequence but with identical molecular weights. These dsRNAs have been separated from each other by denaturation and polyacrylamide gel electrophoresis. The 3' terminal sequences of the major species, LA dsRNA, were determined. Secondary structural analysis supported the presence of two stem and loop structures at the 3' terminus of the LA positive strand. In strain T132B NK-3, both the LA and Lc species are virion encapsidated. Two distinct classes of virions were purified from this strain, each with a different RNA polymerase activity and with distinct protein components. The heavy virions harbored LA dsRNA, whereas the Lc dsRNA species copurified with the light virion peak. Thus, LA and Lc dsRNAs, when present in the same cell, may be separately encapsidated.The killer virus of the yeast Saccharomyces cerevisiae is a model system in which to study the interaction of a multipartite double-stranded (ds) RNA viral genome with its eucaryotic host (for reviews, see references 9, 11, 36, 38

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mentioning

confidence: 65%

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Thiele¹,

Hannig²,

Leibowitz³

1984

Mol. Cell. Biol.

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“…TETRAHYMENA 1VS RNA The 5'-end-labeled IVS was digested under nondenaturing conditions with RNase Ti, which can be used as a probe for guanosine residues in single-stranded regions (27). Cleavage sites were located by denaturing the RNA and subjecting it to electrophoresis on a sequencing gel (Fig.…”

Section: Secondary Structure Of Thementioning

confidence: 99%

“…2 was derived, we mapped additional RNase T1 cleavage sites near the 3' end of the molecule. Sites of cleavage of the double-strand-specific cobra venom RNase (29) and of single-strand-specific S1 nuclease (27) and RNase T2 were similarly determined ( Fig. 1) and are summarized in Fig.…”

Section: Secondary Structure Of Thementioning

confidence: 99%

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Cech¹,

Tanner²,

Tinoco³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

206

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Splicing of the ribosomal RNA precursor of Tetrahymena is an autocatalytic reaction, requiring no enzyme or other protein in vitro. The structure of the intervening sequence (IVS) appears to direct the cleavage/ligation reactions involved in prerRNA splicing and IVS cyclization. We have probed this structure by treating the linear excised IVS RNA under nondenaturing conditions with various single-and double-strand-specific nucleases and then mapping the cleavage sites by using sequencing gel electrophoresis. A computer program was then used to predict the lowest-free-energy secondary structure consistent with the nuclease cleavage data. The resulting structure is appealing in that the ends of the IVS are in proximity; thus, the IVS can help align the adjacent coding regions (exons) for ligation, and IVS cyclization can occur. The Tetrahymena IVS has several sequences in common with those of fungal mitochondrial mRNA and rRNA IMSs, sequences that by genetic analysis are known to be important cisacting elements for splicing of the mitochondrial RNAs. In the predicted structure of the Tetrahymena IVS, these sequences interact in a pairwise manner similar to that postulated for the mitochondrial IVSs. These findings suggest a common origin of some nuclear and mitochondrial introns and common elements in the mechanism of their splicing.Genes coding for rRNA are interrupted by intervening sequences (IVSs) in several species of Tetrahymena and in Physarum polycephalum (1). These split genes are transcribed to give an IVS-containing pre-rRNA which is then spliced in the nucleus (1). In the case of Tetrahymena, the IVS is excised as a unique linear molecule which is subsequently cyclized (2, 3). Both linear and circular forms of the IVS RNA appear to be rapidly degraded in the nucleus and not exported to the cytoplasm (4). The presence of stop codons in all possible reading frames (5, 6) also makes it unlikely that the excised IVS could serve as a mRNA. Splicing of the Tetrahymena pre-rRNA is a novel case of an autocatalytic reaction that, at least in vitro, requires no enzyme or other protein (7). It remains possible that proteins accelerate the rate of splicing or perhaps even regulate the process in vivo.RNA splicing is also required for the expression of genes coding for the large rRNA and various mRNAs in the mitochondria of fungi. Many of the mitochondrial IVSs contain long open reading frames in phase with the upstream exon. The occurrence of trans-recessive splicing-defective mutants that map within three of the cytochrome b gene (cob) introns (8) has led to the proposal of intron-encoded maturases (9). The introns in yeast and Neurospora large mitochondrial rRNA genes also contain long open reading frames (10, 11); these apparently do not encode maturases because splicing of yeast mitochondrial rRNA continues in the absence of protein synthesis (12). The occurrence of nuclear mutants in mitochondrial mRNA and rRNA splicing (13, 14) makes it seem likely that proteins other than the maturases are required...

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“…A variety of chemical and enzymatic probes have been used to investigate the structure of specific RNA molecules in solution (6)(7)(8)(9)(10)(11). The technique that is currently most widely used involves the reaction of end-labeled RNA under nondenaturing conditions, either with an enzyme that specifically cleaves internucleotide bonds in single-stranded or in double-stranded regions, or with a chemical probe that reacts with bases not involved in secondary or tertiary interactions.…”

mentioning

confidence: 99%

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

Inoue¹,

Cech²

1985

Proc. Natl. Acad. Sci. U.S.A.

297

180

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The structure of the intervening sequence (IVS) of the Tetahymena rRNA precursor mediates cleavageligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.Many eukaryotic genes are interrupted by intervening sequences (introns, IVSs), which are removed from transcripts of these genes by the process of RNA splicing (1, 2). We have been studying one example of such an event, the splic- MATERIALS AND METHODSPreparation of RNA. RNA was synthesized by transcription of pIVS11 DNA with purified Escherichia coli RNA polymerase (3) in a solution containing ATP, CTP, and GTP, each at 200 ,uM; 100 ,uM [3H]UTP (New England Nuclear); 250 mM NaCl; 5 mM MgCl2; 1 mM CaCl2; 50 mM Tris Cl, pH 8.0; and 0.07% 2-mercaptoethanol. During transcription at 30°C for 2 hr, RNA splicing and a limited amount of IVS RNA cyclization also occurred. The resultant IVS RNAs were purified by gel electrophoresis (12) followed by gel filtration on Sephadex G-50 and were then ethanol-precipitated in the absence of carrier. For cyclization, L IVS RNA was incubated in 150 mM NaCl/50 mM Na cacodylate, pH 7.5/10 mM MgCl2 at 30°C for 1 hr. The resulting RNA mixture, containing >80% C IVS, was used directly for chemical or enzymatic treatment without further purification.Chemical Modification. The general procedures and precautions described by Peattie (12) were followed. For modification of native C IVS RNA, 0.5 1,u of dimethyl sulfate (Me2SO4) (Aldrich) or 10 1.l of diethyl pyrocarbonate (DEP) (Aldrich) was added to 200 ul of solution containing 0.5-4 pmol of freshly cyclized IVS RNA and incubated at 30°C for 30 min (Me2SO4) or 60 min (DEP). For the "high Mg2+" and "low Mg2+" analyses, the concentrations of NaCl and MgCl2 were adjusted by dilution immediately after the 1-hr cyclization, and the above modification procedures then were ca...

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Structure mapping of 5'-³²P-labeled RNA with S1 nuclease

Cited by 112 publications

References 43 publications

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

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Structure mapping of 5'-32P-labeled RNA with S1 nuclease

Cited by 112 publications

References 43 publications

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences.

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

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Structure mapping of 5'-³²P-labeled RNA with S1 nuclease