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At least 19 recordsLinked to original sources
PubMed1975

A mutant of escherichia coli defective in removing 3' terminal nucleotides from some transfer RNA precursor molecules.

The conversion of precursor RNA into bacteriophage T4 proline and serine transfer RNAs includes two steps for the enzymatic removal of nucleotides from the 3' ends of RNA chains. Neither of these steps occur following infection of a mutant of Escherichia coli that was previously shown to block the suppressor function of T4 serine transfer RNA. Cell-free extracts of this mutant are furthermore deficient in a wild type enzyme activity that removes nucleotides from the 3' ends of one of the RNA chains described above. The relation of this enzyme to other 3' ribonucleases is not known. We subsequently examined the mutant for its ability to support the biosynthesis of other bacteriophage transfer RNAs. In one instance that is analogous to the proline-serine precursor RNA, maturation of the precursor RNA was blocked during infection of mutant cells. In another instance, precursor RNA maturation was normal, even though this involved the removal of 3'nucleotides. These observations point to the possible existence of at least two 3' ribonucleases for the biosynthesis of transfer RNAs.

Base Sequence↗
PubMed1978

Purification and some properties of a specific nuclease which cleaves transfer RNA precursors from the posterior silk gland of Bombyx mori.

A specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5 S precursor RNA to 4.1 S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+. In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0. Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5 S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity. An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5 S precursor RNA.

Ammonium Chloride↗
PubMed1984

Alterations in the maturation and structure of ribosomal precursor RNA in Novikoff hepatoma cells induced by 5-fluorocytidine.

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.

Animals↗
PubMed1985

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors.

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

Base Sequence↗
PubMed1987

The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction.

The secondary structure of the SP6/mouse insulin precursor RNA was determined by psoralen cross-linking experiments. A series of long-range contacts occur within the left half of the pre-mRNA that contains the intervening sequence. Multiple secondary structures for the pre-mRNA exist since some of the interactions share common sites. In splicing buffer but without the splicing extract added, many of these interactions are stable up to at least 50 degrees C. These interactions, however, are dissociated during the in vitro splicing reaction. This dissociation requires ATP and it occurs during the first 30 min. of the splicing reaction. Pre-mRNAs containing psoralen cross-links in different locations within the RNA molecule were purified and used as substrates for in vitro splicing. Psoralen cross-links at any of the double-stranded regions resulted in complete inhibition of the splicing reaction. This indicates that destabilization of the secondary structure of the SP6/mouse insulin pre-mRNA is necessary for in vitro splicing.

Animals↗
PubMed1976

The structural organization of nuclear messenger RNA precursor. I. Reassociation and hybridization properties of double-stranded hairpin-like loops in messenger RNA precursor.

The hybridization and renaturation properties of double-stranded hairpin-like loops isolated from giant nuclear messenger RNA precursor of mouse liver or ascites carcinoma cells were studied. About half of the hairpins in messenger RNA precursor appear to contain similar sequences, as indicated by the very fast kinetics of renaturation of the denatured double-stranded RNA sequences. These sequences have no tissue specificity. About one third of the hairpin sequences can hybridize to messenger RNA. It is suggested that the long hairpins in messenger RNA precursor play the role of sequences separating messenger RNA sequences from non-informative sequences and that these hairpins are recognized by processing enzymes.

Animals↗
PubMed1988

The 9S RNA precursor of Escherichia coli 5S RNA has three structural domains: implications for processing.

The secondary structure of the 9S RNA precursor to ribosomal 5S RNA in Escherichia coli has been determined using chemical reagents and ribonucleases in combination with a reverse transcription procedure. The 9S RNA precursor was generated in vitro by T7 RNA polymerase, and the rrnB operon terminator, T1, was able to terminate the in vitro transcript. The secondary structure model exhibits three structural domains corresponding to a 5' region, a mature region and a terminator region. The mature domain is structurally identical to 5S RNA, and the ribosomal proteins L18 and L25 are able to bind to the precursor. The processing endoribonuclease RNase E cleaves between the structural domains. Moreover, an intramolecular refolding of the nascent transcript must take place if the current view of RNase III processing stems is correct.

Cloning, Molecular↗
PubMed1998

Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1.

Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters. We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co-transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III. RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190-U14 RNAs. Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway. Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14. Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts.

Base Sequence↗
PubMed1995

Genesis of Sindbis virus by in vivo recombination of nonreplicative RNA precursors.

Genetically engineered RNA transcripts coding for various Sindbis virus (SIN) genes were used to study structure and sequence requirements of RNA recombination in BHK cells. Three different groups of RNA transcripts were made: (i) RNAs which retain the ability to replicate and which carry sequences coding for either viral polymerase or viral structural proteins; (ii) RNAs which lack the complete 3' end of the SIN genome and thus are incapable of replicating; and (iii) RNAs which lack the complete 5' end of the SIN genome and also are incapable of replicating. BHK cells were transfected with specific combinations of these precursor RNAs, and virus production and RNA synthetic abilities of the released virus were determined. We demonstrate in vivo generation of infectious SIN by fusion of (i) replicative RNAs to nonreplicative RNAs and (ii) two nonreplicative RNA precursors. Both homologous and nonhomologous types of recombinations were observed. In the homologous type of recombination, a 694-nucleotide overlap at the crossover region of the first pair of precursors resulted in the addition of an A residue converting the UAG stop codon of nonstructural protein P4 to a UAA stop codon. In the nonhomologous type of recombination, the crossover sites contained deletion of up to 76 nucleotides from one of the precursors and complete preservation of junction sequence from the other precursor. This is also the first report that a cytoplasmic RNA virus can be generated from biologically nonreplicative RNA precursors. These results have implications for initiation of viral RNA synthesis and recombination between RNA viral genomes in general. We favor template switching as a mechanism for the fusion events described here and suggest inclusion of polymerase scanning of diverse nonreplicative RNAs as an inherent feature of the copy choice model of RNA recombination. Very importantly, the facile nature of RNA recombination occurring between nonreplicative RNA precursors should speed up the production and analysis of targeted mutants of SIN and possibly other RNA viruses.

Animals↗
PubMed1987

Structure of msDNA from Myxococcus xanthus: evidence for a long, self-annealing RNA precursor for the covalently linked, branched RNA.

The branched RNA (msdRNA) of M. xanthus consists of 77 bases. The 20th rG residue is linked to the 5' end of msDNA, consisting of 162 bases, by a 2', 5' phosphodiester linkage. The msdRNA coding region is located on the chromosome in the opposite orientation to the msDNA coding region, with the 3' ends overlapping by eight bases. S1 nuclease mapping experiments indicate that the primary product of msdRNA is much longer at both the 5' and 3' ends (approximately 375 bases). Because of homologous sequences upstream of the msdRNA and msDNA coding regions, the precursor RNA molecule is considered to form an extremely stable stem-and-loop structure (delta G = -210 kcal). We propose a novel mechanism of DNA synthesis in which the stem-and-loop structure serves as a primer as well as a template to form the branched RNA-linked msDNA.

Base Sequence↗
PubMed1983

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.

Adenosine Triphosphate↗
PubMed1981

The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena.

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.

Animals↗
PubMed1981

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease.

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.

Animals↗
PubMed1980

Transcription pattern of in vivo-labeled late simian virus 40 RNA: evidence that 16S and 19S mRNA's are derived from distinct precursor RNA populations.

The biosynthesis of the two major simian virus 40 mRNA molecules (19S mRNA and 16S mRNA) made at late times in the infective cycle was reinvestigated. By using a modified S1 nuclease technique, we were able to differentiate between pulse-labeled RNA precursor and the spliced mRNA. During a 5-min pulse-labeling with [3H]uridine in vivo, only precursor RNA molecules were detected. Experimental results with polyadenylic acid-selected 5-min pulse-labeled RNA are consistent with the notion that simian virus 40 late RNA can be polyadenylated before final splicing. Finally, 19S mRNA was spliced much more rapidly and appeared more quickly in the cytoplasm than 16S mRNA. Nevertheless, approximately one-half the precursor molecules were destined to become 16S mRNA. Thus, for at least these two viral mRNA's derived from a common transcription unit, the rate of splicing and the rate of nuclear exist are not major determinants of relative mRNA abundance.

Nucleic Acid Precursors↗
PubMed1986

Evidence that the methylation inhibitor cycloleucine causes accumulation of a discrete ribosomal RNA precursor in hamster mitochondria.

A novel RNA fraction, 'Cy RNA,' that accumulates in mitochondria when hamster cells are treated with the methylation inhibitor cycloleucine, has been characterized by high resolution acrylamide gel electrophoresis and DNA-RNA hybridization. Cy RNA ran in gels as a discrete band, with an apparent chain length of 2 600. It hybridized specifically to restriction fragments containing genes for the mitochondrial ribosomal RNAs. We infer that Cy RNA is a discrete polycistronic ribosomal RNA precursor transcript, whose processing is dependent on normal methylation.

Amino Acids↗
PubMed1991

Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors.

Polyadenylation of messenger RNA precursors requires the nucleotide sequence AAUAAA and two factors: poly(A) polymerase and a specificity factor termed cleavage and polyadenylation factor (CPF). We have purified CPF from calf thymus and from HeLa cells to near homogeneity. Four polypeptides with molecular masses of 160, 100, 73, and 30 kDa cofractionate with CPF activity. Glycerol gradient centrifugation and gel filtration indicate that these four proteins form one large complex with a sedimentation constant of 12 S, a Stokes radius near 100 A, and a native molecular mass near 500 kDa. Purified CPF binds specifically to an RNA that contains the AAUAAA sequence. Mutation of the AAUAAA sequence inhibits CPF binding as well as polyadenylation. Purified CPF contains only trace amounts of RNA and does not react with antibodies against common epitopes of small nuclear ribonucleoprotein particles. Thus, contrary to previous indications, CPF does not appear to be a small nuclear ribonucleoprotein particle.

Animals↗