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PubMed1984

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

Adenoviruses, Human↗
PubMed1985

A multicomponent complex is involved in the splicing of messenger RNA precursors.

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.

Adenosine Triphosphate↗
PubMed1987

Splicing of messenger RNA precursors.

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Animals↗
PubMed1975

A novel function of Escherichia coli transfer RNA nucleotidyltransferase. Biosynthesis of the C-C-A sequence in a phage T4 transfer RNA precursor.

The biosynthesis of the phage T4-coded proline and serine transfer RNA species proceeds through a precursor RNA containing both tRNA sequences. Neither tRNA sequence in the precursor RNA contains the 3'-terminal C-C-A common to all mature tRNAs. Seidman and McClain ((1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1491-1495) have proposed that the C-C-A sequence is added to serine tRNA while it is still part of the large precursor RNA. In the present work, I show that, in vitro, a purified preparation of Escherichia coli tRNA nucleotidyltransferase (EC 2.7.7.25) accurately synthesized 3'-terminal C-C-A in the serine tRNA portion of the precursor RNA. This result establishes a role of tRNA nucleotidyltransferase in the biosynthesis of the phage T4 serine tRNA. The finding that tRNA nucleotidyltransferase utilizes the large precursor RNA as a substrate represents a novel function of the enzyme.

Adenine Nucleotides↗
PubMed1999

SR-related proteins and the processing of messenger RNA precursors.

The processing of messenger RNA precursors (pre-mRNA) to mRNA in metazoans requires a large number of proteins that contain domains rich in alternating arginine and serine residues (RS domains). These include members of the SR family of splicing factors and proteins that are structurally and functionally distinct from the SR family, collectively referred to below as SR-related proteins. Both groups of RS domain proteins function in constitutive and regulated pre-mRNA splicing. Recently, several SR-related proteins have been identified that are associated with the transcriptional machinery. Other SR-related proteins are associated with mRNA 3' end formation and have been implicated in export. We review these findings and evidence that proteins containing RS domains may play a fundamental role in coordinating different steps in the synthesis and processing of pre-mRNA.

Animals↗
PubMed1980

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.

Base Sequence↗
PubMed1994

Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.

Amino Acid Sequence↗
PubMed1990

A retrotransposon 412 insertion within an exon of the Drosophila melanogaster vermilion gene is spliced from the precursor RNA.

Three alleles of the Drosophila melanogaster vermilion (v) gene are suppressed by recessive mutations at the suppressor of sable [su(s)], gene. Previous work has established that these alleles have identical insertions of the 412 retrotransposon in the 5'-untranslated region of the gene. Despite the transposon insertion in an exon, v mutants accumulate trace amounts of apparently wild-type-sized transcripts in a su(s)+ background, and the level of v transcript accumulation is increased by su(s) mutations. Here, we have characterized transcripts from a suppressible v mutant in both su(s)+ and su(s)- backgrounds by S1 nuclease protection experiments and sequence analysis of polymerase chain reaction (PCR) generated cDNA clones. We find that transposon sequences are imprecisely eliminated from v mutant transcripts by splicing at donor and acceptor sites located near the ends of the 412 retrotransposon. Four different 5' donor sites are alternatively spliced to a single 3' acceptor site. The implications of this finding are discussed in relation to possible functions of the su(s)+ gene product.

Animals↗
PubMed1990

Corticotropin-releasing factor differentially regulates anterior and intermediate pituitary lobe proopiomelanocortin gene transcription, nuclear precursor RNA and mature mRNA in vivo.

The proopiomelanocortin (POMC) gene and its peptide products are under complex regulation in the pituitary by multiple hormonal, neurohormonal and neurotransmitter factors. Corticotropin-releasing factor (CRF) stimulates the release of POMC-derived peptides in both anterior and intermediate lobes of the pituitary, while having differential long-term effects on levels of POMC mRNA in the two pituitary lobes in vivo. In the present study, we have analyzed the release of POMC-derived peptides, as well as changes in POMC gene transcription, primary transcript, nuclear mRNA and cytoplasmic mRNA levels following acute and chronic in vivo CRF administration in an attempt to elucidate the mechanism whereby this tissue-specific differential regulation occurs. Subcutaneous injection of CRF led to a substantial increase in plasma adrenocorticotropin, but only a minor sustained rise in plasma alpha-melanocyte-stimulating hormone. CRF was shown to induce rapid, time-dependent increases in POMC gene transcription in both anterior and intermediate pituitary lobes, which were reflected by increases in the level of POMC primary transcript in the nucleus. POMC primary transcript remained 2-fold elevated in the anterior lobe for at least 4 h after a single injection of CRF; in contrast, CRF stimulation of POMC primary transcript in the intermediate lobe was short-lived, and had returned to control values by 60 min after injection. After 7 days of repetitive CRF administration, POMC primary transcript, mature nuclear mRNA and cytoplasmic mRNA were 2.5 to 3.0-fold elevated in the anterior pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1985

The ribonuclease-III-processing site near the 5' end of an RNA precursor of bacteriophage T4 and its effect on termination.

Infection of RNase III- (rnc) Escherichia coli cells with bacteriophage T4 delta 27, a deletion mutant missing seven out of the ten genes in the tRNA transcription unit, results in the accumulation of a tRNA precursor (10.5-S RNA) that contains the sequences of tRNAGln, tRNALeu and species 1 RNA [Pragai and Apirion (1981) J. Mol. Biol. 153, 619-630]. In vitro studies, using partially purified RNase III or cell extracts and 10.5-S RNA as substrate, have revealed a cleavage site at the 5' side of the molecule. A computerized secondary structure suggests that the RNase III cleavage site can be placed in a small bulge which could be part of a duplex structure and is adjacent to A-A-G and its complementary sequence U-U-U in the same relative relationships found for most RNase III cleavage sites were the adjacent sequences are (A-A-G/U-U-C). Under normal processing conditions (presence of RNase III) the 3' end of the processed intermediate precursors, 10.1-S and p2Sp1 RNAs, is C-U-U-(U1-2)-UOH, which is determined by a stem and loop structure that could serve as a rho-independent termination signal site. However, in the absence of RNase III, the accumulated 10.5-S precursor RNA does not terminate at the same site and its 3' end is shifted a few nucleotides downstream. Thus, RNase III, besides playing a role in processing of 10.5-S RNA, also affects the termination of that molecule, even though both sites, the RNase III cleavage site and the termination site, are about 390 nucleotides apart.

Binding Sites↗
PubMed1989

Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus.

A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.

DNA, Bacterial↗
PubMed1989

Splicing of the U6 RNA precursor is impaired in fission yeast pre-mRNA splicing mutants.

U6 RNA is a member of a class of small abundant stable nuclear RNAs that are essential for splicing. In all species examined so far, the U6 RNA is a RNA polymerase III transcript. The U6 gene of the fission yeast Schizosaccharomyces pombe is unusual in that it is interrupted by an intron whose structure is similar to those found in pre-mRNAs. As part of our previous analysis of three S. pombe temperature sensitive pre-mRNA splicing mutants we examined their spliceosomal snRNA content. In contrast to the other snRNAs, the amount of U6 RNA is reduced at the restrictive temperature in all three of the mutants compared to the wild type. To investigate the cause of this reduction we have analyzed the efficiency of splicing of the U6 RNA precursor (U6 pre-RNA) in the pre-mRNA splicing mutants. At the restrictive temperature the ratio of unspliced U6 precursor to mature RNA is elevated in the mutants compared to the wild type grown under identical conditions, indicating a defect in U6 pre-RNA splicing. In this regard, the U6 RNA precursor behaves similarly to pre-mRNAs. Unspliced U6 pre-RNA was also detected in wild type cells under certain growth conditions.

Base Sequence↗
PubMed1988

Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA.

Small nuclear ribonucleoprotein particles (snRNPs) associate to form multi-snRNP complexes during splicing of mRNA precursors. A vast majority of the three snRNPs U4, U5, and U6 are present in a nuclear extract in a single complex, while U1 and U2 snRNPs exist as separate particles. Under conditions optimal for splicing in vitro the U4-U5-U6 (U4/5/6) complex dissociates to release free snRNPs, suggesting that the interactions between its components are dynamic. Several forms of splicing complexes assemble on precursor RNA during splicing in vitro. One of these forms, spliceosome B, contains U2, U4, U5, and U6 snRNPs bound to the precursor RNA. This same set of snRNPs associates efficiently in the absence of precursor RNA during incubation of the extract at high salt concentration. Formation of this U2-U4-U5-U6 (U2/4/5/6) complex, the pseudospliceosome, suggests that the basic structure of the spliceosome is specified by snRNP-snRNP interactions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Cleavage factor II of Saccharomyces cerevisiae contains homologues to subunits of the mammalian Cleavage/ polyadenylation specificity factor and exhibits sequence-specific, ATP-dependent interaction with precursor RNA.

Cleavage of pre-mRNA during 3'-end formation in yeast requires two protein factors, cleavage factor I (CF I) and cleavage factor (CF II). A 5300-fold purification of CF II indicates that four polypeptides of 150, 105, 100, and 90 kDa copurify with CF II activity. The 150-kDa protein is recognized by antibodies against Cft1, the yeast homologue of the 160-kDa subunit of the mammalian cleavage/polyadenylation specificity factor (CPSF). The 100-kDa subunit is identical to Brr5/Ysh1, a yeast protein with striking similarity to the 73-kDa subunit of CPSF. The 105-kDa protein, designated Cft2 (cleavage factor two) exhibits significant homology to the CPSF 100-kDa subunit. Cft2 is cross-linked to pre-mRNA substrate containing the poly(A) site and wild type upstream and downstream flanking sequences, but not to precleaved RNA lacking downstream sequences or to substrate in which the (UA)6 processing signal has been deleted. The specific binding of Cft2 to the RNA substrate is ATP-dependent, in agreement with the requirement of ATP for cleavage. The sequence-specific binding of Cft2 and the similarities of CF II subunits to those of CPSF supports the hypothesis that CF II functions in the cleavage of yeast mRNA 3'-ends in a manner analagous to that of CPSF in the mammalian system. These results provide additional evidence that certain features of the molecular mechanism of mRNA 3'-end formation are conserved between yeast and mammals, but also highlight unexpected differences.

Adenosine Triphosphate↗
PubMed1973

Biosynthesis of transfer RNA: in vitro conversion of transfer RNA precursors from Bombyx mori to 4S RNA by Escherichia coli enzymes.

Evidence for the presence of rapidly labeled short-lived RNA species, presumed to be precursors to transfer RNA (tRNA), in the silk glands of Bombyx mori is presented. These precursors, which migrate between 4S and 5S markers on acrylamide gels, can be converted in vitro into molecules indistinguishable in size from tRNA. The conversion is also catalyzed by a crude enzyme fraction isolated from ribosomes of E. coli. Since a similar enzyme preparation cleaves the suppressor tRNA(Tyr) sequence of E. coli from its precursor molecule, it would appear that the precursor to E. coli tRNA(Tyr) and the precursors to silk-gland tRNAs share common structural features.

Animals↗
PubMed1986

Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.

An SP6/mouse insulin RNA precursor containing two exons and one intron can be spliced in a partially purified nuclear extract isolated from MOPC-315 mouse myeloma cells. We have detected the putative RNA splicing intermediate (intron-3'exon) in a lariat form, the excised intron in a lariat form, and the mRNA spliced product. The in vitro splicing reaction of gel-purified RNA precursors requires ATP and Mg2+ and was accompanied by the formation of a 60-40S ribonucleoprotein complex. The formation of the 60S complex requires ATP. At least two Sm snRNPs containing U1 and U2 RNAs are components of the 60-40S complex. The assemble of those snRNPs occurs early during the splicing reaction and it requires ATP and intron containing pre-mRNAs.

Adenosine Triphosphate↗
PubMed1998

Polyadenylation of stable RNA precursors in vivo.

Polyadenylation at the 3' terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.

Base Sequence↗