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PubMed2006

Human telomerase and Cajal body ribonucleoproteins share a unique specificity of Sm protein association.

Cajal bodies are nuclear structures that host RNA modification and assembly reactions. Some RNAs transit Cajal bodies, while others must concentrate in Cajal bodies to function. Here we report that at least a subfraction of human telomerase RNA and individual resident Cajal body RNAs is associated with Sm proteins. Surprisingly, of seven Sm proteins assembled into a heteroheptameric ring, only a subset copurifies telomerase and Cajal body ribonucleoproteins. We show that a Cajal body RNA localization motif determines this specificity. These discoveries expand the cellular repertoire of Sm protein assemblies and their involvement in ribonucleoprotein localization and function.

Antibodies, Monoclonal↗
PubMed2026

Unveiling m7G modification patterns and causal drivers governing intracranial aneurysm rupture risk through multi-omics validation and m7G-MeRIP-seq profiling.

Intracranial aneurysm (IA) rupture causes severe brain hemorrhage with high mortality, yet its molecular drivers remain unclear and better risk prediction is urgently needed. Using transcriptomics, single-cell analysis, and genetic data, we investigated the role of N7-methylguanosine (m7G) RNA modification in IA. We identified distinct m7G modification patterns, validated their methylation features in patient samples, and incorporated these patterns into a machine learning-based rupture prediction model. The presence and characteristics of m7G patterns significantly improved model performance, achieving high predictive accuracy across three independent cohorts (AUC 0.91-0.95). Genetic analyses further identified three causal m7G-related genes (NSUN2, IFIT5, SNUPN), and laboratory experiments confirmed their altered expression and methylation in ruptured aneurysms. Overall, our findings demonstrate that m7G modifications play a key role in IA rupture. The validated prediction model offers strong clinical potential for rupture risk assessment, and the identified genes represent promising therapeutic targets.

Humans↗
PubMed1998

A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein.

Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein-RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.

Binding Sites↗
PubMed2005

RNA damage in human atherosclerosis: pathophysiological significance and implications for gene expression studies.

RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. Here we summarize recent evidence that loss of RNA integrity and 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) oxidative RNA modifications frequently occur in advanced human atherosclerotic plaques. At least two features of advanced human plaques, namely oxidative stress and intraplaque hemorrhage followed by iron deposition, may be involved in the process of RNA degradation. Although speculative, RNA damage may lead to destabilization and rupture of atherosclerotic plaques by interfering with protein synthesis and stimulation of cell death. Moreover, RNA damage may affect in vitro transcript quantification, thereby influencing data from gene expression studies.

Animals↗
PubMed2026

New insights on Plasmodium gene expression from direct RNA sequencing.

Oxford Nanopore Technology (ONT) direct RNA sequencing enables the sequencing of native RNA molecules without cDNA conversion. The long-read approach captures full-length reads spanning entire genes and has transformed the study of gene expression in Plasmodium parasites by enabling analysis of untranslated regions, isoforms, and alternative splicing. In addition, ONT provides unique insights into non-coding RNAs, RNA modifications, and polyadenylated tail dynamics, which are expanding our understanding of post-transcriptional regulation in Plasmodium, including processes beyond translational repression in gametocytes and sporozoites. Here, we discuss the past and future applications of direct RNA sequencing in Plasmodium research and highlight its advantages, limitations, and future prospects.

Oxford Nanopore Technology↗
PubMed2006

Genetic medicines: treatment strategies for hereditary disorders.

The treatment of the more than 1,800 known monogenic hereditary disorders will depend on the development of 'genetic medicines' - therapies that use the transfer of DNA and/or RNA to modify gene expression to correct or compensate for an abnormal phenotype. Strategies include the use of somatic stem cells, gene transfer, RNA modification and, in the future, embryonic stem cells. Despite the efficacy of these technologies in treating experimental models of hereditary disorders, applying them successfully in the clinic is a great challenge, which will only be overcome by expending considerable intellectual and economic resources, and by solving societal concerns about modifications of the human genetic repertoire.

Gene Transfer Techniques↗
PubMed1991

[Complementary addressed alkylation of 16S rRNA of Escherichia coli by 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligodeoxyribonucleotides. V. Study of the factors affecting selectivity of modification].

We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined. The temperature rise from 20 to 40 degrees C brought about a 1.5-fold increase in the relative extent of modification at the target site 771-781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782-789 fragment of 16S rRNA (reagent concentration is 1 x 10(-6) M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1 x 10(-7) M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold) drop of the level of the 16S rRNA alkylation was observed.

Alkylation↗
PubMed1999

Site-specific labeling of RNA with fluorophores and other structural probes.

Site-specific probes provide a powerful tool for structure and function studies of nucleic acids, especially in elucidating tertiary structures of large ribozymes and other folded RNA molecules. Among many types of extrinsic labels, fluorophores are most attractive because they can provide structural information at millisecond time resolution, thus allowing real-time observation of structural transition during biological function. Methods for introducing fluorophores in RNA molecules are summarized here. These methods are robust and readily applicable to the labeling of other types of probes. However, as each case of RNA modification is unique, fine tuning of the general methodology is beneficial.

Amines↗
PubMed1995

Oxidative base damage in RNA detected by reverse transcriptase.

Oxidative base damage in DNA and metabolic defects in the recognition and removal of such damage play important roles in mutagenesis and human disease. The extent to which cellular RNA is a substrate for oxidative damage and the possible biological consequences of RNA base oxidation, however, remain largely unexplored. Since oxidatively modified RNA may contribute to the high mutability of retroviral genomic DNA, we have been interested in developing methods for the sequence specific detection of such damage. We show here that a primer extension assay using AMV reverse transcriptase (RT) can be used to reveal oxidatively damaged sites in RNA. This finding extends the currently known range of RNA modifications detectable with AMV reverse transcriptase. Analogous assays using DNA polymerases to detect base damage in DNA substrates appear to be restricted to lesions at thymine. Oxidative base damage in the absence of any detectable chain breaks was produced by dye photosensitization of RNA. Six out of 20 dyes examined were capable of producing RT detectable lesions. RT stops were seen predominantly at purines, although many pyrimidine sites were also detected. Dye specific photofootprints revealed by RT analysis suggests differential dye binding to the RNA substrate. Some of the photoreactive dyes described here may have potential utility in RNA structural analysis, particularly in the identification of stem-loop regions in complex RNAs.

Base Sequence↗
PubMed2000

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon.

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42781bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT-PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

Algal Proteins↗
PubMed1998

Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme.

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

Genetic Engineering↗
PubMed1999

Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method.

Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly. Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion. A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues. These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions. The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified. By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA. An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.

Escherichia coli↗
PubMed2005

Detection of the common RNA nucleoside pseudouridine in mixtures of oligonucleotides by mass spectrometry.

Pseudouridine, an isomer of uridine, is probably the most common of many posttranscriptional RNA modifications found in nature. Although mass spectrometry has become widely used in the characterization of modified nucleic acids, its application to the recognition and sequence placement of pseudouridine has not been straightforward, particularly in the case of complex mixtures such as those resulting from selective enzymatic hydrolysis of RNA into oligonucleotides. We report results of a study of the characteristic dissociation reactions of pseudouridine-containing oligonucleotides following ionization by electrospray and use of those pathways in an LC/MS-based method applicable to direct analysis of RNase digests of RNA. As a consequence of the C-C (rather than C-N) glycosidic bond of pseudouridine, the otherwise common dissociation paths involving base loss do not occur, resulting in characteristic formation of a set of low-mass negative ions containing the intact glycosidic bond (m/z 225, 207, 189, 165, 164, 139), which permit recognition of pseudouridine-containing oligonucleotides. Those components can subsequently be subjected to sequence analysis by MS/MS, in which enhancement of selective sequence-determining ions (a-, w-, y-types), and absence of a - base ions, are observed at the site of pseudouridylation. Also, selected reaction pathways can be monitored in the LC/MS/MS analysis that are indicative of pseudouridine at the 5' terminus (m/z 225 --> 165), internal positions (m/z 207 --> 164), and in the RNase T1-derived product Psi pGp (m/z 668 --> 207) arising from the RNA sequence ...G Psi G... These procedures can be effectively integrated into an existing suite of LC/ESI-MS-based methods designed for the analysis of posttranscriptionally modified sites in RNA.

Base Sequence↗
PubMed2000

Catalytic nucleic acids: from lab to applications.

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.

Animals↗
PubMed2004

Structures of two new "minimalist" modified nucleosides from archaeal tRNA.

The wyeosine (or wye) family of tricyclic ribonucleosides from archaeal and eukaryal tRNA(Phe) constitutes one of the most complex and interesting series of posttranscriptional RNA modifications, and has been the object of numerous studies of their chemical and biological synthesis and distribution. We report the structures of two minimally elaborated wye derivatives from archaea, raising the known number of wye nucleosides to eight: 3,4-dihydro-6-methyl-3-beta-d-ribofuranosyl-9H-imidazo[1,2-a]purine-9-one (symbol imG-14), and 3,4-dihydro-6,7-dimethyl-3-beta-d-ribofuranosyl-9H-imidazo[1,2-a]purine-9-one (symbol imG2). Structures were determined primarily by mass spectrometry, and confirmed by comparison of physicochemical properties with those of chemically synthesized nucleosides. The nucleosides contain no amino acid side chains at C-7 (1H-imidazo[1,2-a]purine nomenclature) and are the only wye derivatives not methylated at N-4. These features suggest a minimal role for wye methyl groups and side chains in maintenance of anticodon stem-loop structures, and support the concept that archaeal tRNA nucleoside modification motifs are generally simpler than those of their counterparts in eukarya and bacteria.

Archaea↗
PubMed1988

Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA.

Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3'-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3'-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.

Animals↗
PubMed2002

Bioinformatics classification and functional analysis of PhoH homologs.

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.

Bacteria↗