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PubMedDate not supplied

Detection of specific RNA sequences in yeast by in situ colony hybridization.

Recently a convenient method for detection of specific RNA sequences in bacteria has been developed but the original protocol was inapplicable to microorganisms with a rigid cell wall. Here we report a modification of the RNA colony hybridization for use with yeast. The modified method includes the following consecutive procedures: a) treatment of the yeast colonies on the membrane filter with 10% SDS at 65% C for 30 min; b) treatment of the same filter with 3 x SSC, 10% formaldehyde at 65 degrees C for 30 min; c) hybridization with 32P-labelled oligonucleotide (or DNA) specific for the RNA sequence of interest. The intensity of the radioactive signals thus obtained is comparable with that of the E. coli colonies.

Cell Wall↗

PubMed1975

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.

Amanitins↗

PubMed2000

Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain.

Prader-Willi syndrome is a complex neurodevelopmental disorder caused by the inactivation or deletion of imprinted, paternally expressed genes in chromosome band 15q11.2. We report the identification and characterization of PWCR1, a novel imprinted gene within that region, and its mouse orthologue, Pwcr1, which was mapped to the conserved syntenic region on mouse chromosome 7. Expressed only from the paternal allele, both genes require the imprinting-center regulatory element for expression and are transcribed from the same strand. They are intronless and do not appear to encode a protein product. High human/mouse sequence similarity (87% identity) is limited to a 99-bp region called "HMCR" (for "human-mouse conserved region"). The HMCR sequence has features of a C/D box small nucleolar RNA (snoRNA) and is represented in an abundant small transcript in both species. Located in nucleoli, snoRNAs serve as methylation guidance RNAs in the modification of ribosomal RNA and other small nuclear RNAs. In addition to the nonpolyadenylated small RNAs, larger polyadenylated PWCR1 transcripts are found in most human tissues, whereas expression of any Pwcr1 RNAs is limited to mouse brain. Genomic sequence analysis reveals the presence of multiple copies of PWCR1 and Pwcr1 that are organized within local tandem-repeat clusters. On a multispecies Southern blot, hybridization to an HMCR probe encoding the putative snoRNA is limited to mammals.

Alleles↗

PubMedDate not supplied

[Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit].

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

Hepacivirus↗

PubMed1993

Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases.

A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin.

Affinity Labels↗

PubMed1980

T4 RNA ligase as a nucleic acid synthesis and modification reagent.

Oligodeoxyribonucleotides corresponding to portions of the recognition sequence and analogues thereof of the Eco RI restriction endonuclease have been synthesized using T4 RNA ligase. The successive addition of deoxyribonucleoside-3',5'-bisphosphates to preformed deoxyoligomers allowed stepwise oligodeoxyribonucleotide synthesis. Single strand deoxyoligomers were also joined to one another by the enzyme. In addition, biotin, and fluorophore tetramethylrhodamine, and hexylamine have been added to RNA via an ATP-independent RNA ligase reaction using their ADP adducts as substrates. When the beta-substituent on ADP is a good leaving group, e.g. p-nitrophenol or 4-methylumbelliferol, the RNA product is the 2'-(3')-cyclicphosphate derivative.

DNA Restriction Enzymes↗

PubMed1989

U2 small nuclear RNP assembly in vitro.

Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell S100 fraction resulted in the formation of ribonucleoprotein complexes. In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease. These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins. In contrast, the particles that formed in the absence of ATP did not have these properties. ATP analogs with non-hydrolyzable beta-gamma bonds did not substitute for ATP in U2 snRNP assembly. Additional experiments with a mutant U2 RNA confirmed that nucleotides 154-167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins. Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions.

Adenosine Triphosphate↗

PubMed1994

Formation of pseudouridine in U5 small nuclear RNA.

The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

Base Sequence↗

PubMed2001

Molecular tryst peeping: detection of interactions between nonlabeled nucleic acids by fluorescence resonance energy transfer.

We have developed a new method for monitoring the interactions between nonlabeled RNAs that involves detection of fluorescence resonance energy transfer (FRET) between two DNA probes with different fluorescent label. The sequences of the probes are complementary to those of the RNAs. In this study, we examined the interaction between a portion of the LTR RNA of HIV-1 and the corresponding antisense RNA. The antisense RNA was designed not to bind to the fluorescent DNA without prior hybridization to the target RNA. A mixture of RNAs and DNA probes with fluorescent labels was fractionated by electrophoresis on a nondenaturing polyacrylamide gel and then the gel was analyzed with a fluorescence imaging analyzer. FRET was observed only in the presence of target RNA, antisense RNA, and both of the fluorescent DNA probes. This strategy should be useful for the detection of interactions between nucleic acids that cannot be subjected to chemical modification, such as RNA transcripts inside cells.

Base Sequence↗

PubMed1999

Barley yellow dwarf virus RNA requires a cap-independent translation sequence because it lacks a 5' cap.

A 3' translation enhancer (3'TE) sequence that facilitates cap-independent translation is located near the 3' end of barley yellow dwarf luteovirus RNA. Here, we show that the 3'TE is required for translation of the viral genome and thus for viral replication. Antisense inhibition showed that the 3'TE has significant secondary structure and is required for translation of virion RNA from infected plants and uncapped genomic transcripts but not for translation of capped transcripts. Direct end-labeling of RNAs verified the absence of a 5' modification on virion RNA. Thus barley yellow dwarf virus differs from related viruses by having neither a genome-linked protein nor a 5' cap.

Enhancer Elements, Genetic↗

PubMed1980

The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies.

The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.

Amino Acid Sequence↗

PubMed2004

A computational search for box C/D snoRNA genes in the Drosophila melanogaster genome.

MOTIVATION: In eukaryotes, the family of non-coding RNA genes includes a number of genes encoding small nucleolar RNAs (mainly C/D and H/ACA snoRNAs), which act as guides in the maturation or post-transcriptional modifications of target RNA molecules. Since in Drosophila melanogaster (Dm) only few examples of snoRNAs have been identified so far by cDNA libraries screening, integration of the molecular data with in silico identification of these types of genes could throw light on their organization in the Dm genome. RESULTS: We have performed a computational screening of the Dm genome for C/D snoRNA genes, followed by experimental validation of the putative candidates. Few of the 26 confirmed snoRNAs had been recognized by cDNA library analysis. Organization of the Dm genome was also found to be more variegated than previously suspected, with snoRNA genes nested in both the introns and exons of protein-coding genes. This finding suggests that the presence of additional mechanisms of snoRNA biogenesis based on the alternative production of overlapping mRNA/snoRNA molecules. AVAILABILITY: Additional information is available at http://www.bioinformatica.unito.it/bioinformatics/snoRNAs.

Animals↗

PubMed2005

Conserved spacing between the box C/D and C'/D' RNPs of the archaeal box C/D sRNP complex is required for efficient 2'-O-methylation of target RNAs.

RNA-guided nucleotide modification complexes direct the post-transcriptional nucleotide modification of both archaeal and eukaryotic RNAs. We have previously demonstrated that efficient 2'-O-methylation activity guided by an in vitro reconstituted archaeal box C/D sRNP requires juxtaposed box C/D and C'/D' RNP complexes. In these experiments, we investigate the importance of spatially positioning the box C/D and C'/D' RNPs within the sRNP complex for nucleotide modification. Initial sequence analysis of 245 archaeal box C/D sRNAs from both Eukyarchaeota and Crenarchaeota kingdoms revealed highly conserved spacing between the box C/D and C'/D' RNA motifs. Distances between boxes C to D' and C' to D (D' and D spacers, respectively) exhibit highly constrained lengths of 12 nucleotides (nt). Methanocaldococcus jannaschii sR8 sRNA, a model box C/D sRNA with D and D' spacers of 12 nt, was mutated to alter the distance between the two RNA motifs. sRNAs with longer or shorter spacer regions could still form sRNPs by associating with box C/D core proteins, L7, Nop56/58, and fibrillarin, comparable to wild-type sR8. However, these reconstituted box C/D sRNP complexes were severely deficient in methylation activity. Alteration of the D and D' spacer lengths disrupted the guided methylation activity of both the box C/D and C'/D' RNP complexes. When only one spacer region was altered, methylation activity of the corresponding RNP was lost. Collectively, these results demonstrate the importance of box C/D and C'/D' RNP positioning for preservation of critical inter-RNP interactions required for efficient box C/D sRNP-guided nucleotide methylation.

Archaeal Proteins↗

PubMedDate not supplied

Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification.

Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor represents a unique posttranslational modification that is ubiquitously present in eukaryotic cells and archaebacteria. Specific inhibition of deoxyhypusine synthase leads to growth arrest and cell death. The precise cellular function of eIF-5A and the physiological significance of hypusine modification are not clear. Although the methionyl-puromycin synthesis has been suggested to be the functional assay for eIF-5A activity in vitro, the role of eIF-5A in protein synthesis has not been established. Recent studies have suggested that eIF-5A may be the cellular target of the human immunodeficiency virus type 1 Rev and human T cell leukemia virus type 1 Rex proteins. Motif analysis suggested that eIF-5A resembles a bimodular RNA-binding protein in that it contains a stretch of basic amino acids clustered at the N-terminal region and a leucine-rich stretch at the C-terminal region. Using Rev target RNA, RRE, as a model, we tested the hypothesis that eIF-5A may be an RNA-binding protein. We found that both deoxyhypusine and hypusine-containing eIF-5A can bind to the 252-nt RRE RNA, as determined by a gel mobility shift assay. In contrast, the unmodified eIF-5A precursor cannot. Deoxyhypusine-containing eIF-5A, but not its precursor, could also cause supershift of the Rev stem-loop IIB RRE complex. Preliminary studies also indicated that eIF-5A can bind to RNA such as U6 snRNA and that deoxyhypusine modification appears to be required for the binding. The ability of eIF-5A to directly interact with RNA suggests that deoxyhypusine formation of eIF-5A may be related to its role in RNA processing and protein synthesis. Our study also suggests the possibility of using a gel mobility shift assay for eIF-5A-RNA binding as a functional assay for deoxyhypusine and hypusine formation.

Gene Products, rev↗

PubMed1976

Molecular modifications associated with Aging of globin messenger RNA in vitro.

Using polyacrylamide gel elution-electrophoresis in aqueous medium, highly purified rabbit globin mRNA can be fractionated into several populations of molecules differing by their mean poly(A) content. Both alpha and beta globin mRNA are heterogenous with respect to their electrophoretic mobilities. With the conditions used no separation of alpha and beta globin mRNA occurs during electrophoresis. From the specific radioactivity distribution in the different mRNA fractions one can conclude that the polyadenylate sequence at the 3' end of globin mRNA molecules becomes shorter with aging. This shortening occurs on alpha as well as beta, globin mRNAs and the extent of heterogeneity in poly(A) content is similar for both globin mRNAs. Furthermore, using two different methods of mRNA fractionation (polyacrylamide gel elution-electrophoresis and elution of poly (U)-Sepharose-bound mRNA at increasing temperatures) it is shown that old mRNA molecules differ from relatively young messages in their ability to direct cell-free globin synthesis. Modifications reducing template activity in vitro thus seem to take place during globin mRNA aging.

Aging↗

PubMed2002

A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains.

Site-specific post-transcriptional conversion of uridines to pseudouridine in ribosomal RNAs and small nuclear RNAs (snRNAs) is directed by guide RNAs which possess the conserved box H and ACA sequence elements and fold into the consensus 'hairpin-hinge-hairpin-tail' secondary structure. Here, we describe an unusual mammalian pseudouridylation guide RNA, called U93, that is composed of two tandemly arranged box H/ACA RNA domains. The U93 RNA therefore carries two H and two ACA box motifs, all of which are essential for accumulation of the full-length RNA. The human U93 RNA accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA. Our results lend further support to the notion that modification of the RNA polymerase II-transcribed spliceosomal snRNAs takes place in Cajal bodies.

Animals↗

PubMed2005

The role of the RNAi machinery in heterochromatin formation.

Considerable effort has been devoted to the characterization of RNA interference (RNAi), a posttranscriptional gene silencing mechanism involving small RNA-containing effector complexes. Recent studies have revealed that components of the RNAi machinery are associated not only with target RNA cleavage and impairment of target RNA translation but also with the formation of heterochromatin. There is increasing evidence that RNA-mediated chromatin modifications play an important role in epigenetic transcriptional gene silencing.

Animals↗

PubMed1982

Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.

The function of lysyl residues of the sigma subunit of the RNA polymerase from Escherichia coli was investigated by chemical modification with trinitrobenzenesulfonic acid (TNBS). Following reaction with TNBS, analysis of the modified sigma indicated that trinitrophenylation was limited to the epsilon-amino groups of lysyl residues. Progressive loss in the activity of sigma followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of sigma activity. Kinetic analysis indicated that one lysyl group is critical for the function of sigma. TNP-sigma was able to form a holoenzyme complex with a binding affinity comparable to that of sigma. Promoter recognition studies were done by using HindIII fragments from T5 DNA. The TNP-sigma core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d(A-T)n and T7 DNA templates. TNP-sigma was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-sigma or sigma using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the conformation of sigma following trinitrophenylation.

DNA-Directed RNA Polymerases↗