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At least 145 records · Page 8Linked to original sources
PubMed2005

Expression of complementary RNA from chloroplast transgenes affects editing efficiency of transgene and endogenous chloroplast transcripts.

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

Chloroplasts↗
PubMed2004

Decoding the genome: a modified view.

Transfer RNA's role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA's ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA's anticodon and those 3'-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U*U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon-codon interactions and expanding others.

Animals↗
PubMed1978

General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation.

A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper.

Escherichia coli↗
PubMed2005

Induced fit of RNA on binding the L7Ae protein to the kink-turn motif.

The kink-turn is a widespread motif in RNA consisting of a three-nucleotide bulge flanked on one side by consecutive A3G mismatches. Important examples are found in the ribosome, U4 RNA, and in snoRNAs involved in RNA modification. The motif is a common protein binding site, and the RNA has been found to adopt a tightly kinked conformation in crystal structures. However, in free solution there is a dynamic exchange between kinked and extended conformations, with the equilibrium driven toward the kinked form by the addition of metal ions. Here we used fluorescence resonance energy transfer (FRET) to show that the L7Ae protein of Archaeoglobus fulgidus binds to RNA containing a kink-turn with nanomolar affinity, and induces folding into the tightly kinked conformation even in the absence of metal ions. Thus this RNA may act as a relatively flexible hinge during RNA folding, until fixed into its ultimate kinked structure by the binding of L7 or related protein.

Archaeal Proteins↗
PubMed2003

RNA-modifying machines in archaea.

It has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Amino Acid Sequence↗
PubMed2006

Structure and function of RNA replication.

Contrary to their host cells, many viruses contain RNA as genetic material and hence encode an RNA-dependent RNA polymerase to replicate their genomes. This review discusses the present status of our knowledge on the structure of these enzymes and the mechanisms of RNA replication. The simplest viruses encode only the catalytic subunit of the replication complex, but other viruses also contribute a variable number of ancillary factors. These and other factors provided by the host cell play roles in the specificity and affinity of template recognition and the assembly of the replication complex. Usually, these host factors are involved in protein synthesis or RNA modification in the host cell, but they play roles in remodeling RNA-RNA, RNA-protein, and protein-protein interactions during virus RNA replication. Furthermore, viruses take advantage of and modify previous cell structural elements, frequently membrane vesicles, for the formation of RNA replication complexes.

Genome, Viral↗
PubMed2026

How Epitranscriptomic Machinery Senses Environmental Cues.

Environmental fluctuations remodel RNA modification landscapes, yet the routes that connect cue detection to writer-eraser-reader control remain dispersed across disciplines. Here, we consolidate upstream mechanisms capable of driving epitranscriptomic change and organize them by response speed. At the fastest proximal level, catalytic output can be modulated through shifts in substrate and cofactor availability, redox and ionic state, temperature, and direct chemical or metal interference with enzyme active sites, although transcriptome-wide RNA readouts may appear later. Over minutes to hours, cue-responsive signaling can reach the machinery through post-translational modification, partner switching, subcellular trafficking, and stress-induced condensates that may gate access to modified transcripts. Across hours to days, regulator abundance and specificity are reshaped by transcriptional programs, translational control, and protein quality-control pathways, enabling adaptation and, in some contexts, persistence. We propose a kinetics-to-sensors approach for interpreting time-resolved epitranscriptomic datasets and prioritizing perturbations that discriminate among candidate upstream inputs. We also outline conceptual gaps and experimental practices needed to establish causal cue-to-mark chains.

Epitranscriptome↗
PubMed2005

Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes.

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.

Animals↗
PubMed1979

[Quantitative cytochemical study of the RNA in tumor cells. II. The characteristics of the cell populatoins].

The quantity of DNA and RNA in tymocytes of AKR mice has been determined by Methylen blue staining. Cell populations of normal and tumor animals were divided into two groups with equal content of DNA to measure RNA content in either of them. The tumor cells had up to 380% RNA, if the normal content be taken for 100%. Dynamics of RNA modifications in cells of normal tymocytes corresponded to the increasing DNA quantity. The RNA quantity in tumor cells increased in the beginning of S-phase.

Animals↗
PubMed2005

Alanine scanning of MS2 coat protein reveals protein-phosphate contacts involved in thermodynamic hot spots.

The co-crystal structure of the MS2 coat protein dimer with its RNA operator reveals eight amino acid side-chains contacting seven of the RNA phosphates. These eight amino acids and five nearby control positions were individually changed to an alanine residue and the binding affinities of the mutant proteins to the RNA were determined. In general, the data agreed well with the crystal structure and previous RNA modification data. Interestingly, amino acid residues that are energetically most important for complex formation cluster in the middle of the RNA binding interface, forming thermodynamic hot spots, and are surrounded by energetically less relevant amino acids. In order to evaluate whether or not a given alanine mutation causes a global change in the RNA-protein interface, the affinities of the mutant proteins to RNAs containing one of 14 backbone modifications spanning the entire interface were determined. In three of six protein mutations tested, thermodynamic coupling between the site of the mutation and RNA groups that can be even more than 16 A away was detected. This suggests that, in some cases, the mutation may subtly alter the entire protein-RNA interface.

Alanine↗
PubMed2006

Microarray analysis of RNA processing and modification.

Most RNAs are processed from precursors by mechanisms that include covalent modifications, as well as the removal of flanking and intervening sequences. Traditional methods to detect RNA processing, such as Northern blotting, reverse-transcribed polymerase chain reaction and primer extension assays, are difficult to apply on a large scale. This chapter outlines several methods for analysis of the processing and modification of RNA using microarrays. These encompass protocols for the application of homemade microarrays and custom-designed commercial inkjet microarrays and are tailored for the large-scale analysis of processing of mRNA, including alternative splicing, as well as for the analysis of processing and modification of noncoding RNA. This chapter also describes practical aspects of microarray design, sample preparation, hybridization, and data analysis.

Animals↗
PubMed1981

Liver and kidney nuclear RNA synthesis and modifications in dimethylnitrosamine-treated rats.

RNA synthesis was measured in nuclei isolated from rat liver and kidney 22 h post injection of 30 mg dimethylnitrosamine/kg body weight. In nuclear preparations were shown by electron microscopy to consist of clean hepatocytes and the liver nuclei showed no apparent necrosis at that time. In vitro RNA synthesis and methylation were proportional to time and nuclear concentration, as well as dependent on exogenous nucleoside triphosphates and S-adenosylmethionine. 60-70% of the in vitro synthesis was inhibited by 1 microgram/ml alpha-amanitin. Total liver nuclear RNA synthesis was increased after dimethylnitrosamine exposure, but, unlike RNA synthesis in nuclei after partial hepatectomy, both alpha-amanitin-sensitive and -resistant synthesis were increased. Differences were found between dimethylnitrosamine-treated liver and kidney nuclear RNA synthesis which was sensitive to inhibition by 1-10 microgram/ml alpha-amanitin, presumably a product of RNA polymerase III. Nuclear RNA methylation with S-adenosylmethionine, which was dependent on new RNA synthesis, differed between dimethylnitrosamine-treated rat liver and kidney nuclei. The endogenous RNA methyl substituents labeled in vitro showed differences in levels of methylation of bases, the 2'-O position of ribose and caps in comparison between control and dimethylnitrosamine-treated nuclei from both liver and kidney. Significant differences were obtained in both nuclear RNA transcription and methylation in vitro between the two tissues in response to pretreatment of the rat in vito dimethylnitrosamine.

Animals↗
PubMed2001

Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae.

To characterize the substrate specificity of the putative RNA:pseudouridine (Psi)-synthase encoded by the Saccharomyces cerevisiae open reading frame (ORF) YGR169c, the corresponding gene was deleted in yeast, and the consequences of the deletion on tRNA and small nuclear RNA modification were tested. The resulting DeltaYGR169c strain showed no detectable growth phenotype, and the only difference in Psi formation in stable cellular RNAs was the absence of Psi at position 31 in cytoplasmic and mitochondrial tRNAs. Complementation of the DeltaYGR169c strain by a plasmid bearing the wild-type YGR169c ORF restored Psi(31) formation in tRNA, whereas a point mutation of the enzyme active site (Asp(168)-->Ala) abolished tRNA:Psi(31)-synthase activity. Moreover, recombinant His(6)-tagged Ygr169 protein produced in Escherichia coli was capable of forming Psi(31) in vitro using tRNAs extracted from the DeltaYGR169c yeast cells as substrates. These results demonstrate that the protein encoded by the S. cerevisiae ORF YGR169c is the Psi-synthase responsible for modification of cytoplasmic and mitochondrial tRNAs at position 31. Because this is the sixth RNA:Psi-synthase characterized thus far in yeast, we propose to rename the corresponding gene PUS6 and the expressed protein Pus6p. Finally, the cellular localization of the green fluorescent protein-tagged Pus6p was studied by functional tests and direct fluorescence microscopy.

Cytoplasm↗
PubMed2026

Interplay between DNA and RNA methylation shapes cancer cell plasticity.

Cellular plasticity refers to the ability of healthy cells to shift between phenotypic states and modify their characteristics to maintain tissue homeostasis and integrity. In the tumor context, cancer stem cells (CSCs) exploit this flexibility to withstand stress, facilitate tumor dissemination, and evade therapeutic interventions. Epigenetic regulation, particularly DNA methylation at CpG sites, is recognized as a well-known driver of tumor plasticity by repressing differentiation programs through modulation of chromatin accessibility. More recently, RNA modifications (epitranscriptomics) have emerged as crucial post-transcriptional regulators of gene expression that shape RNA fate and function. Among these, N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N7-methylguanosine (m7G) contribute to the regulation of cell identity by modulating stemness-differentiation balance, stress adaptation, and epithelial-to-mesenchymal transition (EMT). Notably, dysregulation of both DNA and RNA methylation signatures is frequently observed in tumors, suggesting potential functional interactions between these regulatory layers. Emerging evidence indicates that DNA CpG methylation and RNA methylation pathways may cooperate to influence stemness, survival, and EMT-associated signaling, thereby supporting CSCs' plasticity. Although the molecular mechanisms underlying this crosstalk remain incompletely understood, accumulating studies suggest that DNA and RNA methylation could converge within interconnected regulatory networks that contribute to the control of cancer cell identity. A deeper understanding of these interactions may uncover novel vulnerabilities for targeting tumor plasticity. In this review, we summarize the current knowledge on the interplay between DNA and RNA methylation in regulating tumor plasticity, highlighting emerging mechanistic insights, functional interactions, and potential implications for future epigenetic and epitranscriptomic therapeutic strategies.

Humans↗
PubMed1997

The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.

The guide RNA (gRNA) paradigm states that the uridine (U) insertion/deletion type of RNA editing is mediated by short 3' uridylylated gRNAs that are complementary to specific blocks of mature edited sequence. These gRNAs contain the edited sequence information in the form of guiding purine residues that can base pair with the inserted U's and do not base pair with encoded U's that are to be deleted. The minicircle gRNA genes in trypanosomatids are localized at specific sites within the variable region, with the number and the precise localization of genes also being species-specific. The total number of minicircle sequence classes and thereby minicircle-encoded gRNAs varies greatly between species and even between different strains of the same species, with the greatest number being in the trypanosome species. Several conundrums which appeared to raise problems for the gRNA paradigm arose during comparative analysis of minicircle gRNA gene organization. The solution of these conundrums has led to a better understanding of the function and evolution of this RNA modification phenomenon.

Animals↗
PubMed2006

Mammalian small nucleolar RNAs are mobile genetic elements.

Small nucleolar RNAs (snoRNAs) of the H/ACA box and C/D box categories guide the pseudouridylation and the 2'-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body-specific RNAs (scaRNAs) guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs) characterized by an A-rich tail and an approximately 14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.

Animals↗
PubMed1997

Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure.

Influenza virus transcription and replication is performed by ribonucleoprotein particles (RNPs). They consist of an RNA molecule covered with many copies of nucleoprotein (NP) and carry a trimeric RNA polymerase complex. RNA modification analysis and electron microscopy performed on native RNPs suggest that the polymerase forms a complex with both conserved viral RNA (vRNA) ends, whereas NP binding exposes the RNA bases to the solvent. After chemical removal of the polymerase, the bases at the vRNA extremities become reactive to modification and the vRNPs behave as structures with free ends, as judged from the observation of salt-induced conformational changes by electron microscopy. The vRNA appears to be completely single-stranded in polymerase-free RNPs despite a partial, inverted complementarity of the vRNA ends. The absence of a stable double-stranded panhandle structure in polymerase-free RNPs has important implications for the mechanism of viral transcription and the switch from transcription to replication.

Animals↗
PubMed1999

Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation.

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

Algal Proteins↗