Uridylic Acid Residues Research Articles

A phosphodiesterase from ascites carcinoma Krebs II cells specifically cleaves the bond between VPg and RNA of encephalomyocarditis virus.

The substrate specificity of the "unlinking" enzyme from ascite carcinoma Krebs II cells has been investigated. The enzyme specifically splits the interpolymeric phosphodiester bond between Kp and the 5;-terminal phosphate group of the uridylic acid residue in the Kp-pUpUpGp complex.

Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs.

Accurate 3′ End Processing and Adenylation of Human Signal Recognition Particle RNA and Alu RNA in Vitro

Human signal recognition particle (SRP) RNA is transcribed by RNA polymerase III and terminates with -GUCUCUUUUOH on its 3' end. Our previous studies showed that the three terminal uridylic acid residues of human SRP RNA are post-transcriptionally removed and a single adenylic acid residue is added, resulting in a 3' end sequence of -GUCUCUAOH (Sinha, K. M., Gu, J., Chen, Y., and Reddy, R. (1998) J. Biol. Chem. 273, 6853-6859). In this study we show that the Alu RNA, corresponding to the 5' and 3' ends of SRP RNA, is also accurately processed and adenylated in vitro. Alu RNAs containing 7 or 11 additional nucleotides on the 3' end were accurately processed and then adenylated. Deletion analysis showed that an 87-nucleotide-long motif comprising of the 5' and 3' ends, including stem IV of the Alu RNA, is sufficient and necessary for the 3' end processing and adenylation. A 73-nucleotide-long construct with deletion of stem IV, required for the binding of SRP 9/14-kDa proteins, was neither processed nor adenylated. The adenylated Alu RNA as well as adenylated SRP RNA were bound to the SRP 9/14-kDa heterodimer and were immunoprecipitated by specific antibodies. A significant fraction of SRP RNA in the nucleoli was found to be processed and adenylated. These data are consistent with nascent SRP and/or Alu RNAs first binding to SRP 9/14-kDa protein heterodimer, followed by the removal of extra sequence on the 3' end and then the addition of one adenylic acid residue in the nucleus, before transport into the cytoplasm.

Formation of 2′,3′-Cyclic Phosphates at the 3′ End of Human U6 Small Nuclear RNA in Vitro: IDENTIFICATION OF 2′,3′-CYCLIC PHOSPHATES AT THE 3′ ENDS OF HUMAN SIGNAL RECOGNITION PARTICLE AND MITOCHONDRIAL RNA PROCESSING RNAs

Approximately 90% of human U6 small nuclear RNA (snRNA) contains uridine cyclic phosphate (U>p) at its 3'-end (Lund, E., and Dahlberg, J. E. (1992) Science 255, 327-330). We studied the formation of U>p at the 3' end of human U6 snRNA using an in vitro system where uridylic acid residues are added from UTP precursor and U>p is formed. Analysis of U6 snRNAs with varying number of uridylic acid residues showed that each of these species contains U>p where the phosphate originated from alpha-phosphate of UTP precursor. The cyclic phosphate formation occurred on U6 snRNA in extracts where essential spliceosomal snRNAs were specifically degraded, thereby indicating that U>p formation is not coupled to pre-mRNA splicing. A subpopulation of human signal recognition particle and mitochondrial RNA processing RNAs isolated from HeLa cells also contained cyclic phosphates at their 3' ends. These data suggest that U>p in U6 snRNA is unlikely to be related to its participation in splicing of pre-mRNAs. It appears that cyclic phosphate is an intermediate product in the metabolism of these small RNAs.

Cyclic 2′,3′-Phosphates and Nontemplated Nucleotides at the 3′ End of Spliceosomal U6 Small Nuclear RNA's

Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally. The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development. The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both).

Multiple termination sites in an unlinked 5S rRNA operon in the archaebacterium, Thermococcus celer

The termini of transcripts from an unlinked 5S rRNA operon were analyzed in the archaebacterium, Thermococcus celer. While the sequences of the 5' termini were completely consistent with the presently accepted consensus promoter sequences, putative multiple termination sites were identified which differed significantly from those identified in other organisms, including archaebacteria. Although termination appears to be dependent on a stem/loop structure this structure does not immediately precede a uridylic acid residue cluster. Rather, an adenylic acid residue cluster follows the termination site. The relationship of this termination site structure to those of other archaebacteria is considered.

The capped U6 small nuclear RNA is transcribed by RNA polymerase III.

U6 RNA is an abundant, capped, small nuclear RNA (snRNA) species associated with heterogeneous nuclear ribonucleoproteins in eukaryotic cells. U4 RNA and U6 RNA are hydrogen bonded in a 1:1 ratio in discrete small nuclear ribonucleoprotein particles that are required in pre-mRNA processing. Previous reports have established that the mRNAs and U1 to U5 U-snRNAs are synthesized by RNA polymerase II. Evidence is presented here for synthesis of U6 RNA by RNA polymerase III. The synthesis of U6 RNA in vitro, using Novikoff hepatoma or HeLa whole cell extracts, was not inhibited at low (1 microgram/ml) concentrations of alpha-amanitin, and only 35% inhibition occurred at 10 micrograms/ml concentration. The in vitro synthesized U6 RNA, like other RNA polymerase III transcripts, was associated with La antigen. The U6 RNA synthesized in vitro by the whole cell extracts was capped, but no other internal post-transcriptional modifications were found. Uridylic acid residues were also added post-transcriptionally to the 3'-end of U6 RNA in vitro. U6 RNA, though capped on its 5'-end, is transcribed by RNA polymerase III; this is the first report of a capped RNA molecule synthesized by RNA polymerase III.

Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation.

To characterize the transcriptional organization and regulation of minute virus of mice, an autonomous parvovirus, viral transcriptional complexes were isolated and cleaved with restriction enzymes. The in vivo preinitiated nascent RNA was elongated in vitro in the presence of [alpha-32P]UTP to generate runoff transcripts. The lengths of the runoff transcripts were analyzed by gel electrophoresis under denaturing conditions. On the basis of the map locations of the restriction sites and the lengths of the runoff transcripts, the in vivo initiation sites were determined. Two major initiation sites having similar activities were thus identified at residues 201 +/- 5 and 2005 +/- 5; both of them were preceded by a TATAA sequence. When uncleaved viral transcriptional complexes or isolated nuclei were incubated in vitro in the presence of [alpha-32P]UTP or [alpha-32P]CTP, they synthesized labeled RNA that, as determined by polyacrylamide gel electrophoresis, contained a major band of 142 nucleotides. The RNA of the major band was mapped between the initiation site at residue 201 +/- 5 and residue 342. We noticed the potential of forming two mutually exclusive stem-and-loop structures in the 142-nucleotide RNA; one of them is followed by a string of uridylic acid residues typical of a procaryotic transcription termination signal. We propose that, as in the transcription of simian virus 40, RNA transcription in minute virus of mice may be regulated by attenuation and may involve eucaryotic polymerase B, which can respond to a transcription termination signal similar to that of the procaryotic polymerase.

Effect of 2'-O-methylation on the structure of mammalian 5.8S rRNAs and the 5.8S-28S rRNA junction.

The mammalian 5.8S rRNA contains a partially 2'-O-methylated uridylic acid residue at position 14 which is largely or entirely methylated in the cytoplasm (Nazar, R.N., Sitz, T.O. and Sommers, K.D. (1980) J. Mol. Biol. 142, 117-121). The effect of this methylation on the 5.8S RNA structure and 5.8-28S rRNA junction was investigated using both chemical and physical approaches. Electrophoretic studies indicated that the free 5.8S rRNA can take on at least two different conformations and that the 2'-O-methylation at U14 restricts the molecule to the more hydrodynamically open form. Structural studies using limited pancreatic or T1 ribonuclease digestion indicated that the methylated conformation was more susceptible to digestion, consistent with a more open tertiary structure. Modification-exclusion studies indicated that the first 29 nucleotides at the 5' end and residues 140 through 158 at the 3' end affect the 5.8S-28S rRNA interaction, supporting previous suggestions that the 5.8S RNA interacts with its cognate high molecular weight component through its termini. These results also suggested that the 2'-O-methylated uridylic acid residue plays a role in the 5.8S-28S rRNA interaction and thermal denaturation studies confirmed this by showing that methylation destabilizes the 5.8S-28S rRNA junction. The 5.8-28S rRNA interaction appears to be more complex than previously believed.

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism.

Ehrlich ascites-tumour cells incubated in a mineral medium supplemented with glucose and glutamine intensely incorporated labelled uridine into free nucleotides and RNA. Detailed kinetic studies of the labelling of total acid-soluble pools of uridine and cytidine nucleotides and of RNA under standard conditions and after chase with non-radioactive uridine were carried out. The relative distribution of the radioactivity between the individual uridine phosphates as well as their total cellular contents were also determined under standard and chase conditions. The labelling kinetics of the mononucleotides and RNA by radioactive uridine and orotate were compared, and some turnover parameters were estimated by mathematical analysis of a steady-state model. The following conclusions were drawn. (1) The observed chase effect of addition of non-radioactive uridine is due to a rapid expansion of the acid-soluble uridine nucleotide pool and consequently a severalfold lowering of its specific radioactivity. (2) The free uridine nucleotides are partly separated into a large and a small compartment, labelled preferentially by exogeneous orotate and uridine respectively. (3) The half-life of the mononucleotides, at least those located in the smaller compartment, is a few minutes only, owing to a rapid exchange with the uridylic acid residues of unstable RNA species. (4) This process of reutilization of RNA-degradation products accounts for 85% (a minimum estimate) of the overall turnover of the nucleotide pool in resting cells.

The purification and properties of chicken liver RNase: An enzyme which is useful in distinguishing between cytidylic and uridylic acid residues.

A heat-stable endoribonuclease isolated from chicken liver has been purified to homogeneity as evidenced by the presence of a single protein band upon polyacrylamide gel electrophoresis. The enzyme can, in limit digests of 5 S rRNA and 5.8 S rRNA, dinstinguish between cytidylic and uridylic acids bonds at a ratio of 61:1 and, therefore, may be useful in RNA sequence analysis. The means by which the enzyme hydrolyzes substrate is unusual in that kinetic data do not support a simple formation and breakdown of an enzyme . substrate complex. Rather, the existence of a second complex, consisting of 2 mol of substrate and one of enzyme, derived from the initial enzyme . substrate complex, is postulated. In common with the other endonucleases, enzyme activity is inhibited by free poly(A) or tracts of the polypurine present at the 3'-terminus of RNA. Reversal of inhibition and restoration of activity may be achieved by the addition of low concentrations of spermidine to reaction mixtures.

A new series of RNAs associated with the genome of spleen focus forming virus (SFFV) and poly(A)-containing RNA from SFFV-infected cells.

A series of low molecular weight RNAs (4.5 to 5.5S) as well as other 4 to 7S RNAs were dissociated from genomic RNA of spleen focus forming virus (SFFV) by heating. On two dimensional polyacrylamide gel electrophoresis, this series of RNAs gave a series of more than thirty spots. RNase T1 fingerprints of these spots were identical except for differences in 3'-terminal oligonucleotides, which were mainly due to different numbers of uridylic acid residues, larger RNA-molecules containing poly(U)sequences at their 3'-termini. This series of RNAs is also associated with poly(A)-containing nuclear and cytoplasmic RNAs from SFFV-infected cells.

O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus.

Virion RNA of poliovirus type 1 has been analyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid or alkali and enzymatic degradation lead to the conclusion that the bond is neither a phosphodiester such as nucleotidyl-(P-O)-serine (or threonine) nor a phosphoramidate such as nucleotidyl-(P-N)-amino acid. VPg-RNA can be iodinated by the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] but not by the chloramine-T or lactoperoxidase procedures, an observation suggesting that VPg does not contain accessible tyrosine. However, VPg can be labeled with [3H]tyrosine in vivo. Hydrolysis of VPg-[32P]pUp with 5.6 M HCl at 110 degrees yielded 32P-labeled O4-(3'-phospho-5'-uridylyl)tyrosine that could be cleaved with micrococcal nuclease to O4-[32P]phosphotyrosine and uridine 3'-[32P]phosphate. These data establish that VPg is linked to the poliovirus genome by a bond between the O4 of tyrosine and the 5'-P atom of the terminal uridylic acid residue. The 5' end of polio genome RNA can now be described as VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G.

Studies of low molecular weight RNA from cells infected with adenovirus 2. I. The sequences at the 3' end of VA-RNA I.

VA-RNA I is a low molecular weight RNA produced in large amounts in cells infected with adenoviruses. The 3' terminus of this RNA may represent a transcription termination site. We have demonstrated that this RNA occurs in infected cells in several forms which differ in the number of uridylic acid residues at the 3' ends. The nucleotide sequence of a DNA fragment overlapping the 3' end of VA-RNA I has been determined. The DNA could encode up to 4 uridylic acid residues at the 3' end of the RNA. The DNA sequence shows some similarity to known transcription termination sequences in prokaryotic systems.

Evidence for the Direct Binding of Polyamines to a Ribonuclease that Hydrolyzes Ribonucleic Acid at Uridylic Acid Residues

Abstract The Citrobacter ribonuclease, an enzyme that cleaves preferentially phosphodiester bonds of ribonucleic acid at uridylic acid residues, has been shown to bind the polyamines, putrescine, spermidine, and spermine. The binding of spermidine insures the maintenance of enzyme activity in the presence of the polynucleotide, polyguanylic acid, at a concentration which is ordinarily completely inhibitory. Enzyme activity, if inhibited by polyguanylic acid, could, however, be restored by spermidine, which appears to effect a removal of the polynucleotide from the enzyme surface. The substitution of the polyamine for the polynucleotide was used to obtain two spermidine-bound peptides from tryptic digests of a polyguanylic acid-enzyme complex. Amino acid analysis of the isolated peptides showed that one consisted of a chain having a minimum length of 17 residues but composed of 10 different amino acids. The other had a minimum length of 8 residues and contained only 6 different amino acids. In both cases, the ratio of peptide to polyamine was approximately 1 to 1.

Effect of Polyamines on a Ribonuclease Which Hydrolyzes Ribonucleic Acid at Uridylic Acid Residues

Abstract A heat-stable ribonuclease, isolated from Citrobacter species, catalyzes the conversion of polyuridylic acid to cyclic 2',3'-uridylic acid but does not hydrolyze polycytidylic, polyguanylic, or polyadenylic acids. The enzyme, purified some 1500-fold, migrates as a single band after electrophoresis on polyacrylamide gels and exhibits an absorption maximum and minimum at 281 and 248 nm, respectively. Evidence from studies with synthetic polyribonucleotides and with yeast ribonucleic acid suggests that the nuclease has a distinct predilection for internucleotide bonds containing uridylic acid at either end of the phosphodiester linkage. Enzyme activity is affected profoundly by the polyamines, putrescine and spermidine. Both of these substances as well as the ammonium ion stimulate enzyme activity considerably. In addition, spermidine appears to alter the high degree of specificity exhibited by the enzyme to an activity much more random in nature. Enzyme activity is inhibited by polyguanylic acid and by the double- and triple-stranded complexes, polyadenylate + polyuridylate and polyadenylate + 2 polyuridylates, respectively. The inhibition of enzyme activity induced by these polynucleotides was found to be competitive, and activity could be restored completely by spermidine.

Translation and translocation of defined RNA messengers

A purified system for protein synthesis, derived from Esherichia coli, makes use of small, synthetic mRNA's initiated by the fMet ‡ ‡ Abbreviations used: fMet, N-formylniethionine. As far as possible all other abbreviations follow the recommendations of IUPAC-IUB as published in J. Biol. Chem. (1966) 241, 527. AUGU 3 and AUGU indicate the trinucleoside diphosphate ApUpG followed by 3, 6 or 9 uridylic acid residues; GMP PCP, 5'-guanylylmethylene diphosphonate. codon, AUG, followed by a sequence of 3, 6 or 9 uridylic acid residues. These di-, tri- and tetra-codons direct the binding of fMet- and Phe-tRNA to ribosomes and the synthesis of the corresponding fMet-initiated di-, tri- and tetrapeptides. This system, directing the synthesis of unique and conveniently detected oligopeptide products, has permitted us to correlate the soluble elements required for protein synthesis with specific steps in the translation of initial and succeeding codons. Complete translation of these small mRNA's has certain of the stringent requirements necessary for the accurate cell-free translation of naturally occurring RNA messengers. Thus, initiation factors, fMet-tRNA and GTP must be provided. After recognition of the first codon and formation of the initial complex, one of two transfer factors, in the presence of GTP, catalyzes a binding reaction in which the second (first internal) codon is recognized. This permits formation of the dipeptide, fMet-Phe, but does not permit translation of succeeding codons. A second transfer factor, probably an enzyme, participates in a translocation reaction in which the third codon is made available for translation, apparently displacing the second codon at the recognition site on the ribosome. In addition, the complex formed between peptidyl-tRNA, mRNA and ribosome is stabilized, possibly by translocation of the peptidyl-tRNA from a site of lesser affinity to one of greater affinity on the ribosome. The role of GTP in the translocation reaction will be certain only when it has been uncoupled from its participation in the binding reaction. Once requirements for translation of the third codon have been met no further additions are necessary for elongation of the peptide chain.

Helix-with-loops structure of polynucleotide. I. Poly(C + IC) and poly(C + GU).

Abstract A copolymer of 57% riboinosinic acid plus 43% ribocytidylic acid has been prepared. The ultraviolet absorbances of aqueous solutions of mixtures of this copolymer and polyribocytidylic acid of various mole ratios were measured. The results indicate that, in these aqueous solutions, a “helix-with-loops” structure is formed; this structure has the hypoxantine-cytosine base pair and the unpaired cytidylic acid residues in the loops. A copolymer of 58% riboguanylic acid plus 42% ribouridylic acid has also been prepared. A similar ultraviolet examination was made of a mixture of this copolymer and polyribocytidylic acid; again the formation of a “helix-with-loops” structure was indicated. This structure is considered to have the guanine-cytosine base pair and the uridylic acid residues in the loops.

The action of snake venom phosphodiesterase on liver ribosomal ribonucleic acids

The stepwise hydrolysis of rat-liver ribosomal RNA's (rRNA) with snake venom phosphodiesterase (EC 3.1.4.1) has been studied. The following results have been obtained: 1. 1. Hydrolysis of poly (U), poly (A) and equimolar mixtures of them proceeds to completion and yields pU and pA as the only major products of the reaction. Secondary structure in poly (U) · poly (A) has no detectable influence on the rate and the extent of hydrolysis. The substrate (Ap) nCp is resistant to the action of this enzyme, while (Ap) nC is rapidly degraded to give pA and pC. 2. 2. As shown by agar-gel electrophoresis, the liver rRNA studied is constituted of homogeneous 28-S and 18-S RNA molecules. They display secondary structure transitions typical for native rRNA's. The preparations studied are virtually free from endonucleolytic contaminants. 3. 3. Hydrolysis of rRNA's with venom phosphodiesterase goes to completion and yields pG, pC, pA and pU as the only major products. Analysis by determination of the nucleosides originating from chain terminals during hydrolysis, shows that the endonuclease contaminants in this system may account for no more than 1 to 2 internal breaks per RNA molecule. 4. 4. The molar ratio of the mononucleotides liberated on stepwise hydrolysis of liver rRNA's with venom phosphodiesterase varies with progress of enzymatic degradation. The product obtained at early stages of enzyme action has a higher content of pA and pU and a lower content of pG and pC. The same correlation is observed with 18-S rRNA and partly with 28-S rRNA. It is suggested that in rRNA the segment near the 3′-end of the chain contains more pA and pU and less pG and pC as compared with the segment near the 5′-end of the chain. 5. 5. Labeling in vivo of free uridine 5′-phosphates and cytidine 5′-phosphates with [6- 14C]orotic acid shows with both nucleotides a maximum incorporation at 40 min. Ribosomal RNA's labeled for 90 min in vivo with [6- 14C]orotic acid were analysed by partial degradation with venom phosphodiesterase and determination of the radioactivity of uridylic acid residues. The labeling of 18-S RNA is higher than of 28-S RNA. Enzymatic liberation of labeled uridylic acid from 18-S and 28-S RNA is non-uniform. With both 18-S and 28-S RNA, uridylic acid liberated at the initial stages of enzyme action is more highly labeled than uridylic acid from the remaining RNA fragments.

Gel filtration heterogeneity of Escherichia coli soluble RNA

Gel filtration heterogeneity (Sephadex G100) is observed for sRNA preparations obtained from various strains of Escherichia coli by phenol extraction and elution from DEAE-cellulose using M-sodium chloride. These preparations contain, in addition to the transfer RNA which accounts for 75% of the soluble RNA, ribosomal and messenger-like RNA, both of which are soluble in cold (4°C) M-sodium chloride, plus an RNA containing 122 ± 10% nucleotides per chain having a major nucleotide composition similar to transfer RNA, but unable to accept amino acids under the usual in vitro conditions. Moreover, it is devoid of methylated nucleotides, pseudouridylic acid, reduced pyrimidine nucleotides, and is terminated at each end by a uridylic acid residue, with the 5′ and 3′ termini being phosphorylated and unesterified, respectively.