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PubMed1980

Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method.

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.

Base Sequence↗
PubMed1998

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies.

BACKGROUND: The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms. RESULTS: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern. CONCLUSIONS: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.

Animals↗
PubMedDate not supplied

[RNA synthesis in rat brain during pharmacological modification of norepinephrine metabolism].

Norepinephrine metabolism and nuclear RNA (nRNA) synthesis in the rat brain are found to be conjugated. Under the effect of preparations inducing a disturbance in the norepinephrine (sodium diethyldithiocarbamate and reserpine) its content in the brain tissue lowers and the nRNA synthesis intensity decreases. Accumulation on total resources of norepinephrine in the brain under the effect of ipraside or its synaptic form (melipramine, Lu-5) intensifies the nRNA synthesis.

Animals↗
PubMed2026

The pseudouridine epitranscriptomic landscape of advanced prostate cancer therapeutic resistance identifies TIMM17A as a key player.

BACKGROUND: Resistance to androgen receptor signaling inhibitors (ARSIs) remains a major barrier of advanced prostate cancer (PCa) treatment. While RNA epitranscriptomic modifications are increasingly recognized as key regulators of tumor biology, the role of pseudouridine (Ψ) in therapeutic resistance is largely unexplored. METHODS: A darolutamide-resistant PCa cell model was established and subjected to integrated multi-omics profiling using bulk RNA sequencing and photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq). Differential expression and pseudouridylation analyses were combined to identify Ψ-associated genes. Public datasets validated expression and prognosis. Functional assays including RNA knockdown, cell proliferation, colony formation, and xenograft models were conducted. Single-cell RNA sequencing investigated tumor microenvironment (TME) interactions. RESULTS: We identified extensive transcriptomic and pseudouridylation alterations associated with ARSI resistance, with a significant positive correlation between Ψ modification and mRNA expression. Integrated analysis highlighted a subset of "hyper-up" genes enriched in resistance-related pathways. Thus, TIMM17A was identified as a novel candidate. TIMM17A expression was significantly elevated in PCa and correlated with disease progression and poor prognosis. Experimental validations demonstrated that TIMM17A promoted tumor growth and resistance, while its knockdown restored sensitivity to darolutamide both in vitro and in vivo. Mechanistically, TIMM17A expression may be regulated by PUS1‑mediated pseudouridylation. Single-cell analysis further revealed that TIMM17A is enriched in malignant epithelial cells and associated with enhanced cell-cell communication within the TME. CONCLUSIONS: This study delineates the pseudouridine epitranscriptomic landscape in advanced PCa and identifies TIMM17A as a key mediator of therapeutic resistance. Targeting the Ψ-TIMM17A axis may offer a novel strategy to overcome ARSI resistance.

Advanced prostate cancer↗
PubMed2003

Spore-specific modification of DNA-dependent RNA polymerase alpha subunit in streptomycetes--a new model of transcription regulation.

At the very beginning of spore germination in streptomycetes the full-length alpha subunit of DNA-dependent RNA polymerase is shortened from its C-terminus. The C-terminal domain of the protein is required for binding of DNA and transcription regulators but its regulatory role in streptomycetes was not extensively studied. Comparison of the sequences of E. coli and S. coelicolor RNA polymerase alpha subunit (RNAP alpha) C-terminal domains reveals that the majority of amino acid residues responsible for the interaction with transcription regulators is conserved in both microorganisms. The spore specific modification of streptomycete RNAP alpha could thus have its regulatory role. The nature of the proteolytic enzyme, responsible for the RNAP alpha cleavage is discussed.

DNA-Directed RNA Polymerases↗
PubMedDate not supplied

[Modifications of ribosomes from rat liver with alkylating derivatives of tRNA].

Alkylating analogs of peptidyl-tRNA: N-chloroambucilyl-14C-phenylanalyl-tRNA (1), N-iodoacetyl-14C-phenylalanyl-tRNA (2) and N-bromo-acetyl-14C-phenlalanyl-tRNA (3) were applied for the modification of the peptidyl-transferase center of the 80S ribosomes from rat liver. These analogs, being in the teronary complex poly-U: ribosome : tRNA analog, modified ribosomal proteins and ribosomal RNA. The modification is directed to large ribosomal subunit. It is found, that (1) modifies ribosomal proteins L5, L25, L31 and L32 and (2) modifies ribosomal proteins L4, L6, L10+L11, L13 and L30.

Animals↗
PubMed1976

Location of accessible bases in Escherichia coli formylmethionine transfer RNA as determined by chemical modification.

Chemical modification of Escherichia coli tRNAfMet with 1 M chloroacetaldehyde, pH 5.5-6.0 at 25 degrees C, has been found to result in alteration of six cytidine and five adenosine residues in the molecule. The modified cytidine residues are the same as those previously found to be reactive with sodium bisulfite at pH 6.0. The accessible adenosine residues are A36 in the anticodon, A58 in the T psi C loop, and A73, A74, and A77 in the 3; terminal sequence. No modification of adenosine residues in the dihydrouridine or variable loops or of adenosine residues on the 3' side of the anticodon loop could be detected. Treatment of fMet-tRNAfMet with chloracetaldehyde gave the same pattern of midofication as was observed with deacylated tRNAfMet. Chemical modification of E. coli tRNAfMet with 2 sodium bisulfite, pH 7.0 at 25 degrees C, resulted in selective modification of exposed uridine residues in the tRNA. Only three sites were found to be reactive: U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. The overall pattern of chemical modification of tRNAfMet is very similar to that found by others for yeast tRNAPhe, supporting the idea that many of the tertiary interactions in the two tRNAs are the same. The adenosine residue at position 58 in the center of the T psi C loop of the initiator tRNA shows unusual reactivity, however, being modified by chloroacetaldehyde at the same rate as the 3' terminal adenosine residue. This result is in sharp contrast to the uniform resistance of nucleotides in the T psi C loop of yeast tRNAPhe to chemical modification.

Acetaldehyde↗
PubMed1999

Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

APOBEC-1 Deaminase↗
PubMed1976

The specific role of ribosomal protein S1 in the recognition of native phage RNA.

The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation. The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation and midly unfolded phage RNA (by modification with formaldehyde) are independent of S1. It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA. A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.

Antigen-Antibody Reactions↗
PubMed2005

Hypomodification of transfer RNA in cancer with respect to queuosine.

Queuosine is a highly modified nucleoside analogue of guanosine. It is present only in the first position of anticodon loop of specific tRNA i.e., tRNA(his), tRNA(asp), tRNA(asn) and tRNA(tyr) and post transcriptionally modified with base-for-base exchange of guanine to queuine. The transfer RNA modifying enzyme transfer RNA guanine transglycosylase (TGTase) catalyzes the modification of tRNAs. Transfer RNA is completely modified with respect to queuosine in mature tissue, however modification is often incomplete in mitotically active cells. Hypomodification of transfer RNA is correlated with cell proliferation and malignancy. In the present study queuosine modification of transfer RNA and TGTase activity is compared in normal, Dalton's lymphoma ascites transplanted (DLAT) cancerous and queuine treated DLAT cancerous mouse liver. Transfer RNA of cancerous mouse is hypomodified in terms of queuosine modification. TGTase activity of cancerous mouse is found to decrease to less then half of enzyme activity of normal mouse; suggesting that the enzyme may be responsible for transfer RNA hypomodification. Exogenous treatment of queuine during development of cancer improves the queuosine modification of transfer RNA. The activators NaPP and ATP enhance TGTase activity of normal and DLAT cancerous mouse, where as 7mG inhibits the TGTase activity.

Animals↗
PubMed2001

Alternative ribonucleic acid processing in endocrine systems.

Alternative RNA processing is a mechanism for creation of protein diversity through selective inclusion or exclusion of RNA sequence during posttranscriptional processing. More than one-third of human pre-mRNAs undergo alternative RNA processing modification, making this a ubiquitous biological process. The protein isoforms produced have distinct and sometimes opposite functions, underscoring the importance of this process. This review focuses on important endocrine genes regulated by alternative RNA processing. We discuss how diverse events such as spermatogenesis or GH action are regulated by this process. We focus on several endocrine (calcitonin/calcitonin gene-related peptide) and nonendocrine (Drosophila doublesex and P-element and mouse c-src) examples to highlight recent progress in the elucidation of molecular mechanisms regulating this process. Finally, we outline methods (model systems and techniques) used by investigators in this field to study processing of individual pre-mRNAS:

Alternative Splicing↗
PubMed2001

RNA fragmentation studied in a matrix-assisted laser desorption/ionisation tandem quadrupole/orthogonal time-of-flight mass spectrometer.

We have studied the fragmentation behaviour of short, singly protonated oligoribonucleotides on a MALDI Qq-TOF instrument with the aim of using this instrumental set-up to characterise modifications of RNA molecules. Individual ion species from enzymatically generated mixtures were isolated in one quadrupole and subjected to collision-induced dissociation in a second quadrupole followed by separation of the resulting product ions in an orthogonal time-of-flight mass analyser. Complex spectra were generally observed with nearly all types of cleavages along the phosphodiester backbone and of the N-glycosidic bonds (and combinations of these) occurring, albeit at different relative intensities. The most labile part of the backbone was found to be the 5'-P-O bond, resulting in c- and y-ions. Loss of neutral cytosine and guanine occurred equally often, whereas neutral loss of adenosine was less prevalent. Loss of uracil, either neutral or charged species, was not observed. Because the fragmentation pattern observed here is significantly different from what has been reported for singly protonated oligodeoxyribonucleotides, we suggest that the 2'-substituent in the sugar plays a central role in the fragmentation mechanisms of nucleic acids. Finally, we used the acquired knowledge about oligoribonucleotide fragmentation to characterise an in vivo methylated oligoribonucleotide by tandem mass spectrometry.

Base Sequence↗
PubMed1983

Regulation of ribosomal RNA transcription during differentiation of Acanthamoeba castellanii: a review.

During the cellular differentiation induced by starvation of Acanthamoeba castellanii, the expression of a number of genes is regulated. Evidence is reviewed that at least one of these, the precursor ribosomal RNA transcription unit, is regulated at the level of transcription. The structure of the rRNA transcription unit and of the RNA polymerases responsible for transcription in Acanthamoeba are reviewed. Utilizing an in vitro transcription system constructed from these components, preliminary evidence has been obtained that pre-rRNA gene expression is regulated by a modification of RNA polymerase I that affects the enzyme's ability to participate efficiently in the initiation of transcription. These results are reviewed in relation to other known mechanisms of transcriptional regulation in eukaryotes.

Amoeba↗
PubMed1985

Nucleic acid and protein metabolism in undernutrition and protein deficiency.

This review discusses the metabolism of nucleic acids and proteins in various models of undernutrition in female rats and their neonatal and 21-day-old progeny. Based on the observations noted in our laboratories and those of other investigators, it is concluded that body and organ weights as well as various parameters of cellular growth (DNA, RNA, proteins, amino acids and total nucleotides) fail to increase normally in dietary-insulted animals. Protein and RNA synthesis demonstrate variable responses, leading to the speculation that modulation of mRNA metabolism and of protein synthesis occurs in dietary-restricted rats. These findings are also confirmed by the organ weight to DNA ratios. It is further noted that, despite the increases in protein and RNA synthesis in certain organs, protein and RNA register below-normal values, indicating that their degradation is much faster than their formation. This postulate is supported: by the enhanced activities of acid cathepsin (a protein-degrading enzyme) and of RNAse A (a RNA-degrading enzyme); by the elevated concentrations of circulating amino acids and total nucleotides; as well as by the accelerated excretion of nitrogenous compounds in the urine and feces of dietary-restricted animals. Modifications of RNA turnover are also evident in the tRNA and soluble RNA fractions of the liver of dietary-insulted rats. Studies on brain mRNA translatability have revealed: that food deprivation elicits a shorter species of pre-mRNA via a reduced polynucleotide elongation rate; that not all poly A+ RNA sequences present in control rats occur in dietary-restricted animals; and that the translatability of polymerase II is far lower in dietary-insulted rats. Other investigations on the translatability of liver, brain, kidney, spleen and thymus mRNA have demonstrated changes in mRNA via altered protein synthesis in various organs of dietary-restricted rats. Generation studies have shown that adaptation prevails in the first, second and third generation offspring of dietary-insulted rats, after which all parameters decline in fourth and fifth generation offspring. By reducing the litter size and exchanging the pups of control and dietary-restricted rats during the lactation period, partial restoration of the cellular growth of different organs is effected with the exception of the brain, in which damage is irreversible.

Animals↗
PubMed1972

Nucleotide-dependent inactivation of RNA polymerase from Bacillus brevis.

RNA polymerase has been purified from vegetative cells of Bacillus brevis and resolved into "core" enzyme and sigma factor. The purified enzyme is rapidly inactivated by incubation at low temperatures in the presence of 1-2 mM ATP, dATP, or NAD(+), while other nucleotides at this concentration have little or no effect. Inactivation is not accompanied by the incorporation of an adenylyl or phosphoryl moiety into RNA polymerase; nevertheless, it is essentially irreversible. DNA, high concentrations of glycerol, as well as low concentrations (1 mM) of orthophosphate protect RNA polymerase from the nucleotide-dependent inactivation.A similar inactivation of RNA polymerase in the presence of ATP is observed with crude preparations from Bacillus subtilis and Bacillus polymyxa. This phenomenon may represent a novel mode of regulation of transcription that does not involve a covalent modification of RNA polymerase or its interaction with other protein factors, but rather is due to a structural transition to an inactive form induced by small molecules.

Adenosine Diphosphate↗
PubMed1997

Ssd1p of Saccharomyces cerevisiae associates with RNA.

The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4, slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.

Amino Acid Sequence↗
PubMed2006

Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin.

The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.

Amino Acid Sequence↗
PubMed2006

The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase.

BACKGROUND: Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT. RESULTS: As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases. CONCLUSION: Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).

Escherichia coli Proteins↗