Seryl transfer RNA changes during estrogen-induced phosvitin synthesis and a unique seryl transfer RNA modification.
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Animals↗
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Results for “RNA modifications”
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Absence of post-transcriptional RNA modifications of BCL10 in human malignant mesothelioma and colorectal cancer.
The BCL10 gene, located at 1p22, has been implicated in a number of human malignancies, including malignant mesotheliomas (MMs) and colorectal carcinomas. Subsequent reports, however, have revealed an absence of BCL10 mutations in genomic DNA from such tumors. It has been proposed that some abnormalities of this gene may be found only in RNA and not in genomic DNA, suggesting that BCL10 may be mutated post-transcriptionally, rather than at the genomic level. To explore this possibility, we performed SSCP mutation analysis and direct sequencing of cDNA from 17 MM cell lines displaying LOH in 1p22, 12 MM tumor specimens, and 11 colon carcinoma cell lines. SSCP revealed several different band shifts in these samples. The nucleotide changes observed in the cDNA samples were also seen in matched genomic DNA and corresponded to known polymorphisms in the general population. Thus, we conclude the BCL10 mutations are absent at the cDNA level, and that this gene does not undergo "molecular misreading." Since BCL10 also does not possess mutations at the genomic DNA level, it can be ruled out as a gene involved in the pathogenesis of MM and colorectal cancer.
Adaptor Proteins, Signal Transducing↗
Transfer RNA modifications and synthesis in animal cells.
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Animals↗
Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting.
Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.
Anticodon↗
Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro.
The modification patterns of in vitro transcripts of two yeast Saccharomyces cerevisiae tRNAs (tRNAPheand tRNAAsp) and one archaeal Haloferax volcanii tRNA (tRNAIle) were investigated in the cell-free extract of Pyrococcus furiosus supplemented with S -adenosyl-l-methionine (AdoMet). The results indicate that the enzymatic formation of 11 distinct modified nucleotides corresponding to 12 enzymatic activities can be detected in vitro. They correspond to the formation of pseudouridines (Psi) at positions 39 and 55, 2' -O- ribose methylations at positions 6 (Am) and 56 (Cm), base methylations at positions 10 (m2G), 26 (m22G), 37 (m1G), 49 (m5C), 54 (m5U) and 58 (m1A) and both the deamination and methylation of adenosine into m1I at position 57. Most of the detected modified nucleotides are common modifications found in other phylogenetic groups, while Am6, Cm56and m1I57are specific modifications found exclusively in Archaea. It is also shown that the enzymatic formation of m5C49, m5U54, Psi55and m1I57does not depend on the three-dimensional architecture of the tRNA substrate, since these modi-fications also occur in fragmented tRNAs as substrate.
Base Sequence↗
Transfer RNA modification status influences retroviral ribosomal frameshifting.
The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3' side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsn without the Q base reduced the stimulatory effect of tRNAPhe without the Y base, whereas addition of tRNAAsn with the Q base did not alter the stimulatory effect. The addition of tRNAAsn without the Q base and tRNAPhe with the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon.
Animals↗
Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene.
Full expression of the virulence genes of Shigella flexneri requires the presence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2-methylthio-N6-isopentenyladenosine (ms2i6A37, adjacent to and 3' of the anticodon)]. The synthesis of these two nucleosides depends on the products of the tgt and miaA genes respectively. We have shown that the intracellular concentration of the virulence-related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of VirF in the tgt and the miaA mutants suppressed the less virulent (tgt) or the avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulence phenotypes at 30 degrees C, but forced synthesis of VirF at 30 degrees C induced the virulence phenotype at this low temperature. In addition, removal of the known gene silencer H-NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a virulent phenotype at 30 degrees C. Conversely, decreased expression of VirF at 37 degrees C induced by the addition of novobiocin, a known inhibitor of gyrase, led to an avirulent phenotype. We conclude that tRNA modification, temperature and superhelicity have the same target - the expression of VirF - to influence the expression of the central regulatory gene virB and thereby the virulence of Shigella. These results further strengthen the suggestion that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacterial translational machinery in the regulation of the expression of virulence genes which appears here quantitatively as important as the well-established regulation on the transcriptional level.
Antigens, Bacterial↗
Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.
tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.
Amino Acid Sequence↗
A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase.
The modified nucleoside 1-methyladenosine (m(1)A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m(1)A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m(1)A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m(1)I57). In yeast, the formation of m(1)A58 is catalysed by the essential tRNA (m(1)A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m(1)A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m(1)A MTases is discussed in the context of different hypotheses of the tree of life.
Amino Acid Sequence↗
RNA editing: exploring one mode with apolipoprotein B mRNA.
RNA editing is a newly described genetic phenomenon. It encompasses widely different molecular mechanisms and events. According to the specific RNA modification, RNA editing can be broadly classified into six major types. Type II RNA editing occurs in plants and mammals; it consists predominantly in cytidine to uridine conversions resulting from deamination/transamination or transglycosylation, although in plants other mechanisms have not been excluded. Apolipoprotein B mRNA editing is the only well-documented editing phenomenon in mammals. It is an intranuclear event that occurs posttranscriptionally, coincident with splicing and polyadenylation. Recent observations indicate that the tissue- and sequence-specific process is mediated by an enzyme that has separate domains for editing and sequence recognition. The presence of apolipoprotein B mRNA editing activity in tissues that do not produce the protein suggests that other RNAs may be edited and RNA editing may be a genetic phenomenon of general biological importance to the cell.
APOBEC-1 Deaminase↗
Kinetoplastid RNA editing does not require the terminal 3' hydroxyl of guide RNA, but modifications to the guide RNA terminus can inhibit in vitro U insertion.
During RNA editing in kinetoplastid parasites, trans-acting guide RNAs (gRNAs) direct the insertion and deletion of U residues at precise sites in mitochondrial pre-mRNAs. We show here that some modifications to the 3' terminal ribose of gRNA inhibit its ability to direct in vitro U insertion. However, we found that gRNAs lacking this moiety in some circumstances support in vitro editing. Thus, the 3' OH is not required. Inhibition resulting from gRNA modification can be overcome by increasing the gRNA-pre-mRNA base-pairing potential upstream of the editing site, suggesting an importance for this interaction to productive processing.
Animals↗
In vitro evidence that eukaryotic ribosomal RNA transcription is regulated by modification of RNA polymerase I.
We have utilized a cell-free transcription system from Acanthamoeba castellanii to test the functional activity of RNA polymerase I and transcription initiation factor I (TIF-I) during developmental down regulation of rRNA transcription. The results strongly suggest that rRNA transcription is regulated by modification, probably covalent, of RNA polymerase I: (1) The level of activity of TIF-I in extracts from transcriptionally active and inactive cells is constant. (2) The number of RNA polymerase I molecules in transcriptionally active and inactive cells is also constant. (3) In contrast, though the specific activity of polymerase I on damaged templates remains constant, both crude and purified polymerase I from inactive cells have lost the ability to participate in faithful initiation of rRNA transcription. (4) Polymerase I purified from transcriptionally active cells has the same subunit architecture as enzyme from inactive cells. However, the latter is heat denatured 5 times faster than the active polymerase.
Amoeba↗
Principles of selective inactivation of a viral genome. Comparative kinetic study of modification of the viral RNA and model protein with oligoaziridines.
Comparative kinetic analysis of inactivation of bacteriophage MS2 infectivity and aminoalkylation of a model protein (trypsin inhibitor) with oligoaziridines was performed in order to evaluate the selectivity of viral RNA modification with oligocationic reagents. The transition from ethyleneimine monomer to di-, tri-, and tetramer leads to a sharp increase in the rate constant of infectivity inactivation, whereas the rate constant of protein modification changes insignificantly. The selectivity coefficient of the phage RNA aminoalkylation relative to trypsin inhibitor modification increases in this series by more than an order of magnitude. This effect is probably associated with the strengthening of the reagent binding to the nucleic acid, which implies a reaction mechanism that involves the formation of a reactive intermediate. The latter might be an electrostatic complex of the oligocationic reagent and RNA, the only polyanion in the virion. A pronounced decrease in the rate constant of infectivity inactivation in the presence of multiply charged anions (in phosphate buffer) and a biogenic polyamine (spermine) favors this hypothesis. Increasing the reaction temperature increases the rate constant of infectivity inactivation and decreases selectivity of the viral RNA modification.
Alkylation↗
Characterization of METTL3/14-mediated m6A modification in human transcriptome using Nanopore direct RNA sequencing.
Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.
Humans↗
Structural modifications of RNA influence the 5' exoribonucleolytic hydrolysis by XRN1 and HKE1 of Saccharomyces cerevisiae.
Two 5' exoribonucleases, XRN1 and HKE1, of Saccharomyces cerevisiae have been found to have very important cellular roles, XRN1 playing a key role in mRNA turnover and HKE1 in pre-rRNA processing. Here, an analysis of strong secondary structures in RNA that cause blocks or stalls (accumulation of RNA fragments that are shortened from the 5' end to the site of the secondary structure insertion) in the processive exoribonucleolytic hydrolysis reactions is reported. With both enzymes, oligo(G) tracts of lengths 18, 16, and 9 stall quite effectively, and the stalls are close to the start of the oligo(G) stretch. Two strong stem-loop structures cause measurable but low-level stalls with both enzymes. If the stem-loop structure is placed close to the 5' end of the RNA, substantial inhibition of overall RNA hydrolysis occurs with HKE1 and less, but measurable, inhibition with XRN1. RNA structural modification caused by protein complexing has been investigated by using poly(A) binding protein. The hydrolysis of poly(A) by XRN1 is inhibited by poly(A) binding protein, while HKE1 activity is not affected.
Base Sequence↗
Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification.
Forms of RNA polymerase I prepared from growing or encysted Acanthamoeba are equal in the ability to transcribe poly(dl:dC). Polymerase from cysts, whose rRNA genes are transcriptionally inactive, is unable to utilize the rDNA promoter in vitro, whereas the transcription initiation factor from cysts is fully able to bind the promoter and direct transcription. Footprinting shows that polymerase from cysts is functionally inactive because of its inability to bind to the promoter. The polymerase footprint moves downstream the appropriate number of base pairs upon various nucleotide additions, without affecting the factor footprint. These results support our hypothesis that rRNA synthesis in eukaryotes is regulated by polymerase I modification and not by alterations to additional DNA-binding proteins.
Amoeba↗
Meta-PseU: A meta-classifier for robust prediction of RNA pseudouridine modification sites from long sequences.
BACKGROUND AND OBJECTIVES: Pseudouridine (Ψ) represents one of the most abundant and conserved RNA modifications. Ψ provides an additional hydrogen-bond donor that enhances RNA structural stability and modulates translation. It participates in diverse biological processes, including RNA-protein interactions, splicing, translational control, and stress responses. Aberrant pseudouridylation is implicated in cancer, neurodegenerative disorders, and autoimmune diseases. Despite its biological importance, experimental identification of Ψ sites remains time-consuming and costly, limiting the feasibility of transcriptome-wide profiling. Computational approaches have therefore become essential complements to experimental techniques. However, state-of-the-art machine-learning and deep-learning predictors often suffer from limited generalizability due to small training datasets. To overcome these issues, we aim at constructing new long-sequence datasets and developing a novel Ψ site predictor. METHODS: New long-sequence datasets were constructed as benchmarks for RNA Ψ-site prediction. The Ψ modification sites in RMBase 3.0 were mapped to the reference genomes across three species of human, mouse, and yeast, and the RNA sequences with a length of 201 were generated by extending the upstream and downstream from the mapped, central sites. To eliminate sequence redundancy, the sequences were clustered using CD-HIT with a 70% sequence identity threshold. We developed Meta-PseU, a logistic regression-based meta-classifier that considered 118 machine learning and deep learning classifiers. The datasets and programs are freely accessible at https://github.com/kuratahiroyuki/MetaPseU. RESULTS: By optimizing model configuration, we proposed the Meta-PseU model stacking 32 machine learning and deep learning classifiers out of 118 classifiers. Meta-PseU substantially improved model generalizability, overcoming a key limitation of existing approaches. It greatly outperformed state-of-the-art predictors and achieved increasing accuracy with increasing sequence length. CONCLUSIONS: Long-sequence datasets were newly constructed as benchmarks for RNA Ψ-site prediction. Meta-PseU offers a new framework for robust Ψ-site identification by using long sequences.
Pseudouridine↗
Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.
The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.
Base Sequence↗