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At least 127 records · Page 7Linked to original sources
PubMed1989

Glucocorticoid-induced stimulation of ribosomal gene transcription in rat hepatoma cells is mediated by modification of RNA polymerase I or an associated factor.

An in vitro transcription system was developed from H411EC3 (H4) hepatoma cells, which mimics the in vivo up-regulation by glucocorticoid hormones on ribosomal RNA (rRNA) synthesis. Ribosomal DNA (rDNA) transcription in extracts derived from H4 cells grown in the presence of 100 nM triamcinolone acetonide was 4- to 5-fold greater than that in extracts derived from cells grown in the absence of glucocorticoid. This effect was not a general stimulation by the steroid, as RNA polymerase II transcription of the metallothionein-1 gene which lacked a glucocorticoid responsive element was unaffected. The increased transcription in hormone-treated extracts was also independent of differential ribonuclease activities or inhibitors as ascertained by the inclusion of ribonuclease inhibitor and mixing experiments, respectively. Chromatography of H4 cell extracts on heparin-sepharose followed by transcription complementation analysis, showed that the hormone-induced stimulatory activity eluted with the fraction (TFIA) which contains RNA polymerase I (Pol I). Immunoblot analysis with specific anti-Pol I antibody showed similar subunit profiles in the absence and presence of the hormone. The presence of a Pol I enhancer element in addition to the rDNA promoter did not further modify the glucocorticoid-induced transcription. These results indicate that the glucocorticoid-mediated effects could be observed in cell extracts which accurately initiate transcription of cloned rat rDNA. Moreover, the alterations of rDNA transcription by the hormone is effected by a factor which elutes with fraction TFIA.

Animals↗
PubMed1975

Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG- are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article).

Cations, Divalent↗
PubMedDate not supplied

[Modification of RNA polymerase from Escherichia coli by fluoresceinbimercuriacetate].

RNA polymerase was modified by fluoresceinbimercuriacetate (FMA) at SH-groups. One molecule of the enzyme (M. W. 500,000) was shown to bind two molecules of FMA. The first molecule reacts with the most reactive SH-groups causing 50%-inhibition of RNA synthesis. The binding of the second one results in 100%-inactivation of the enzyme. Considerable changes in spectral properties of the dye are observed when it interacts with RNA polymerase.

Binding Sites↗
PubMed2004

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

Animals↗
PubMed1999

Triplex formation of chemically modified homopyrimidine oligonucleotides: thermodynamic and kinetic studies.

We have investigated effects of chemical modifications of a third strand on the thermodynamic and kinetic properties of the triplex formation between a 23-bp duplex and each of four kinds of 15-mer chemically modified third strands using isothermal titration calorimetry and interaction analysis system. The chemical modifications of the third strand included one base modification, with replacement of thymine by uracil; two sugar moiety modifications, RNA and 2'-O-methyl-RNA; and one phosphate backbone modification, with replacement of phosphodiester by phosphorothioate backbone. The thermodynamic and kinetic parameters obtained were similar in magnitude at room temperature for the triplex formation with the base-modified and the sugar-modified third strands. By contrast, binding constant for the triplex formation with the third strand containing phosphorothioate backbone was much smaller by a factor of 10 than that for the other triplex formations. Kinetic analyses have also demonstrated that the third strand containing phosphorothioate backbone was much slower in the association step and much faster in the dissociation step than the other third strands, which resulted in the much smaller binding constant. The reason for the instability of the triplex with the third strand containing phosphorothioate backbone will be discussed. We conclude that, at least in the triplex formation with the chemically modified third strands studied in the present work, the modification of phosphate backbone of the third strand produces more significant effect on the triplex formation than the modifications of base and sugar moiety.

Base Sequence↗
PubMed2006

Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR.

Buffers↗
PubMed2026

A pan-cancer analysis of MEX3D in human tumors.

BACKGROUND: MEX3D, a member of the MEX3 RNA-binding protein family, has emerged as a potential regulatory molecule in cancer. However, its role across different tumor types remains largely unexplored. METHODS: We conducted a pan-cancer analysis of MEX3D using transcriptomic and proteomic data from the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Clinical Proteomic Tumor Analysis Consortium (CPTAC). Expression patterns, clinical correlations, survival outcomes, genetic alterations, RNA modification associations, immune infiltration, and functional enrichment were systematically evaluated. RESULTS: MEX3D was significantly dysregulated in numerous cancers at both mRNA and protein levels. Its expression correlated with tumor stage in ACC, LIHC, OV, SKCM, and THCA. Elevated MEX3D expression was associated with poor overall survival (OS) and disease-specific survival (DSS) in multiple malignancies, including ACC, LGG, LUAD, and MESO. Genetic alteration analysis revealed frequent amplifications and mutations, particularly in SARC and OV. MEX3D was positively correlated with RNA modification-related genes (m1A, m5C, m6A) and immune regulatory genes such as CD276, TGFB1, VEGFA, and ICOSLG. Additionally, MEX3D expression showed significant associations with tumor mutational burden (TMB), microsatellite instability (MSI), and cancer-associated fibroblast infiltration. Functional enrichment analyses indicated that MEX3D-related genes are involved in reproductive cellular processes, RNA binding, the Hippo signaling pathway, and microRNA-related oncogenic pathways. CONCLUSION: This pan-cancer analysis highlights the heterogeneous expression and cancer-specific prognostic significance of MEX3D. MEX3D is associated with immune infiltration, immune regulatory genes, RNA modification-related genes, TMB/MSI, and pathways involved in gene regulation and tumor progression. These findings suggest that MEX3D may participate in cancer-specific post-transcriptional and microenvironmental regulatory networks.

Biomarker↗
PubMed2002

Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component.

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification-dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

Fluorescent Antibody Technique↗
PubMed2005

Identification of RNA editing sites in the SNP database.

The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing calls for a careful distinction between inherited SNPs and somatic DNA and RNA modifications. In order to demonstrate that some of the SNP database records are actually somatic modification, we focus on one type of these modifications, namely A-to-I RNA editing, and present evidence for hundreds of dbSNP records that are actually editing sites. We provide a list of 102 RNA editing sites previously annotated in dbSNP database as SNPs, and experimentally validate seven of these. Interestingly, we show how dbSNP can serve as a starting point to look for new editing sites. Our results, for this particular type of RNA editing, demonstrate the need for a careful analysis of SNP databases in light of the increasing recognition of the significance of somatic sequence modifications.

Base Sequence↗
PubMed2004

RNA modulation, repair and remodeling by splice switching oligonucleotides.

Targeting splicing by antisense oligonucleotides allows RNA modifications that are not possible with RNA interference or other antisense techniques that destine the RNA for destruction. By changing the ratio of naturally occurring splice variants the expression of mRNA is modulated. By preventing the use of an aberrant splice site created by a mutation and enforcing re-selection of correct splice sites the RNA is repaired. Antisense induced skipping of the exon that carries a nonsense mutation remodels the mRNA and restores the reading frame of the defective protein. All of the above approaches have clinical applications. Modulation of splice variants is particularly important since close to 60% of all genes code for alternatively spliced pre-mRNA.

Alternative Splicing↗
PubMed2026

m6A-Mediated epitranscriptomic control of mitochondrial dysfunction in neurodegeneration.

Mitochondrial dysfunction is a common pathology of neurodegenerative diseases, which contributes to neuronal vulnerability via excessive oxidative stress, impaired bioenergetics, and dysregulated apoptosis. Emerging studies highlighted the critical role of epitranscriptomic RNA modifications, particularly N6-methyladenosine (m6A), in mitochondrial gene expression regulation and cellular stress responses. m6A modifications are installed by methyltransferases ("writers," METTL3/METTL14), recognized by reader proteins (YTH domain family proteins, IGF2BPs), and removed by demethylases ("erasers," FTO, ALKBH5), collectively orchestrating mRNA splicing, localization, stability, and translation. Recent evidence demonstrates that m6A modifications modulate both nuclear-encoded and mitochondrially encoded transcripts and regulate key mitochondrial processes, including fission/fusion dynamics, oxidative phosphorylation, mitophagy, and apoptosis. Dysregulation of m6A machinery disrupts mitochondrial homeostasis, exacerbates oxidative stress and neuroinflammation, and promotes neuronal loss. Importantly, pharmacological or genetic modulation of m6A regulators can restore mitochondrial function, inhibit caspase activation, and dampen pro-inflammatory signaling, underscoring their therapeutic potential. This review consolidates current insights into mitochondrial epitranscriptomics, emphasizing how m6A modifications act as central regulators of mitochondrial stress responses and neurodegeneration.

Humans↗
PubMed2026

Dogme: a nextflow pipeline for reprocessing nanopore RNA and DNA modifications.

MOTIVATION: Oxford Nanopore (ONT) sequencing allows for the direct detection of RNA and DNA modifications from unamplified nucleic acids, which is a significant advantage over other platforms. However, the rapid updates to ONT basecalling models and the evolving landscape of computational tools for modification detection bring about challenges for reproducible and standardized analyses. To address these challenges, we developed Dogme to automate basecalling, alignment, modification detection, and transcript quantification. Dogme automates the reprocessing of ONT POD5 files by integrating basecalling using Dorado, read mapping using minimap2 and subsequent analysis steps such as running modkit. The pipeline supports three major types of sequencing data-direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA). Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2'-O-methylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA. RESULTS: We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected 96 603 m6A, 43 476 m5C, 8829 inosine, 10 055 pseudouridine, and 30 320 Nm sites in three biological replicates. The pipeline produced reproducible modification profiles and transcript expression levels across replicates, demonstrating its utility for integrative long-read transcriptomic and epigenomic analyses. AVAILABILITY AND IMPLEMENTATION: Dogme is implemented in Nextflow and is freely available under the MIT license at https://github.com/mortazavilab/dogme, with documentation provided for installation and usage.

RNA↗
PubMed2026

The N6-methyladenosine reader IGF2BP2 in T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) represents a highly heterogeneous and aggressive lymphoid neoplasm that lacks pathogenic biomarkers of RNA modification with therapeutic potential. IGF2BP2 is recognized as an N6-methyladenosine reader critically involved in oncogenesis. In this study, we observed consistently high expression of IGF2BP2 across common nodal PTCL subtypes in 3 independent external cohorts, which was further confirmed in our RNA-sequencing (RNA-seq) data set of 196 patients with newly diagnosed PTCL. Both in vitro and in vivo, IGF2BP2 promoted tumor cell growth and inhibited CD8+ T-cell infiltration within the tumor microenvironment. Mechanistically, IGF2BP2 bound to endosome-related genes (RAB4, VPS35, RAB9, and STAM) to maintain their stability, which resulted in enhanced endocytic activity and increased internalization of membrane proteins, and ultimately induced tumor cell proliferation and inhibition of CD8+ T-cell-mediated tumor cytotoxicity. The relationship between IGF2BP2 and endocytosis-associated genes was confirmed using RNA-seq data from patients with PTCL. IGF2BP2 as an upstream regulator of both tumor growth and immune suppression was further demonstrated in patient-derived xenograft models and a coculture system established using tumor samples from patients with PTCL and peripheral blood mononuclear cells. Notably, therapeutic targeting of IGF2BP2 with CWI1-2 suppressed endocytosis and impeded tumor growth in both cell lines and patient-derived xenograft models. Collectively, our findings highlight IGF2BP2 as a clinically relevant oncogenic driver in PTCL that integrates tumor-intrinsic growth signals with immune evasion through endocytosis-centered regulation, providing a novel therapeutic rationale for RNA modification-based strategies that concurrently target tumor cells and the tumor microenvironment.

Humans↗
PubMed2005

The snRNP 15.5K protein folds its cognate K-turn RNA: a combined theoretical and biochemical study.

The human 15.5K protein binds to the 5' stem-loop of U4 snRNA, promotes the assembly of the spliceosomal U4/U6 snRNP, and is required for the recruitment of the 61K protein and the 20/60/90K protein complex to the U4 snRNA. In the crystallographic structure of the 15.5K-U4 snRNA complex, the conformation of the RNA corresponds to the family of kink-turn (K-turn) structural motifs. We simulated the complex and the free RNA, showing how the protein binding and the intrinsic flexibility contribute to the RNA folding process. We found that the RNA is significantly more flexible in the absence of the 15.5K protein. Conformational transitions such as the interconversion between alternative purine stacking schemes, the loss of G-A base pairs, and the opening of the K-turn occur only in the free RNA. Furthermore, the stability of one canonical G-C base pair is influenced both by the binding of the 15.5K protein and the nature of the adjacent structural element in the RNA. We performed chemical RNA modification experiments and observed that the free RNA lacks secondary structure elements, a result in excellent agreement with the simulations. Based on these observations, we propose a protein-assisted RNA folding mechanism in which the RNA intrinsic flexibility functions as a catalyst.

Base Sequence↗
PubMed2006

Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

Archaea↗
PubMed2025

N6-methyladenosine modification of the subgroup J avian leukosis viral RNAs attenuates host innate immunity via MDA5 signaling.

Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host's innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3' untranslated region (3'UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-β production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-β activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3'UTR, exhibited reduced replication capacity and elevated IFN-β expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host's innate immune response through MDA5 signaling pathway.

Animals↗
PubMed2000

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

Base Sequence↗