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At least 19 recordsLinked to original sources
PubMed1992

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs.

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.

Base Composition↗
PubMed2024

Advances in solid-state NMR methods for studying RNA structures and dynamics.

Ribonucleic acid (RNA) structures and dynamics play a crucial role in elucidating RNA functions and facilitating the design of drugs targeting RNA and RNA-protein complexes. However, obtaining RNA structures using conventional biophysical techniques, such as X-ray crystallography and solution nuclear magnetic resonance (NMR), presents challenges due to the inherent flexibility and susceptibility to degradation of RNA. In recent years, solid-state NMR (SSNMR) has rapidly emerged as a promising alternative technique for characterizing RNA structure and dynamics. SSNMR has several distinct advantages, including flexibility in sample states, the ability to capture dynamic features of RNA in solid form, and suitability to character RNAs in various sizes. Recent decade witnessed the growth of 1H-detected SSNMR methods on RNA, which targeted elucidating RNA topology and base pair dynamics in solid state. They have been applied to determine the topology of RNA segment in human immunodeficiency virus (HIV) genome and the base pair dynamics of riboswitch RNA. These advancements have expanded the utility of SSNMR techniques within the RNA research field. This review provides a comprehensive discussion of recent progress in 1H-detected SSNMR investigations into RNA structure and dynamics. We focus on the established 1H-detected SSNMR methods, sample preparation protocols, and the implementation of rapid data acquisition approaches.

Dynamics↗
PubMed2026

RNA structures regulate norovirus life cycle and enable rational attenuation in vivo.

Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell chemical probing, revealing conserved motifs that regulate viral function. Targeted disruption of specific structural elements reduces viral replication in cell culture, modulates translation in cis, and decreases viral RNA levels in animal infection models. These findings enabled the rational design of a genetically stable, attenuated virus that elicits protective immunity and limits viral replication upon secondary challenge. Together, this work uncovers essential roles for RNA structure in norovirus biology and establishes a generalizable framework for RNA structure-guided design of antiviral vaccines and therapeutics.

RNA structure↗
PubMed1991

Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element.

To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2. Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly. Splicing complexes form at 23 degrees C in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15 degrees C, a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome. In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature. We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures. Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.

Base Sequence↗
PubMed1991

Determination of an RNA structure involved in splicing inhibition of a muscle-specific exon.

We have investigated the RNA structure of the region surrounding the muscle-specific exon 6B of the chicken beta-tropomyosin gene. We have used a variety of chemical and enzymatic probes: dimethylsulfate, N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p-tolu enesulfonate) , RNase T1 and RNase V1. Lead acetate was also used to obtain some information on the tertiary structure of this region. Probing the wild-type sequence suggests a model involving one-stem and three-stem-loop structures in and around this exon. Two of these, hairpin I and stem III, have previously been implicated in repression of splicing of the intron following exon 6B in a HeLa nuclear extract. Stem I includes sequences at the beginning of exon 6B and stem III results from interaction of the intron upstream from exon 6B with sequences in the middle of the intron downstream from this exon (the intron whose splicing is repressed). Neither stem I nor stem III directly involves the consensus sequences (5' splice site, branch-point, 3' splice site) of the repressed intron. Probing RNAs that are derepressed for splicing of this intron show that there are structural changes around the 5' splice site and branch-point sequence that correlate with the derepression. This is true, despite the fact that the derepressed RNAs are altered in a region far from these consensus sequences. The most striking structural correlation with splicing capacity of the intron downstream from exon 6B is seen by probing with lead acetate. Lead ions cut RNA at specific residues; these sites are very sensitive to RNA tertiary structure. Repressed and derepressed RNAs show entirely different cleavage patterns after incubation with lead acetate. Remarkably, hybridizing a derepressed RNA with an RNA comprising the ascending arm of stem III not only re-establishes repression, but also converts the pattern of susceptibility to attack by lead ions over the whole molecule. We suggest that RNA conformation plays a role in keeping exon 6B from being spliced into non-muscle cell mRNA.

Animals↗
PubMed2025

Precursor RNA structural patterns at SF3B1 mutation sensitive cryptic 3' splice sites.

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3' splice site selection. The SF3B1 K700E mutation (lysine to glutamic acid) is common in myelodysplastic syndrome and other blood disorders. SF3B1 K700E mutants utilize novel cryptic 3' splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 cryptic 3' splice junctions with significantly altered use in SF3B1 K700E cells, termed SF3B1-sensitive cryptic 3' splice sites, and 2800 cryptic 3' splice sites used in SF3B1 wild-type, termed SF3B1-resistant. We find that SF3B1-sensitive cryptic 3' splice sites are embedded in extended polypyrimidine tracts. Furthermore, canonical splice sites paired to SF3B1-sensitive cryptic 3' splice sites are significantly weaker than canonical 3' splice sites paired to SF3B1-resistant cryptic 3' splice sites. We test whether SF3B1-sensitive splice sites are structurally different from SF3B1-resistant 3' splice sites using chemical probing. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3' splice sites is similar. However, the magnitude of accessibility differences is less in paired SF3B1-sensitive splice sites than in paired SF3B1-mutant splice sites. Additionally, SF3B1-sensitive splice junctions are more flexible than SF3B1-resistant junctions. Our results suggest that SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3' splice site recognition in the presence of SF3B1 mutation.

RNA Splicing Factors↗
PubMed1991

Phylogenetic comparative analysis of RNA structure on Macintosh computers.

A Macintosh Hypertalk program (Hypercard 'stack') for use in phylogenetic comparative analysis of RNA structure is described. The program identifies covariations and compensatory changes in RNA sequence alignments, for use in the construction of secondary structure models or the identification of tertiary interactions. The results of an analysis are presented either as a list of positions in the alignment which covary, or as a 2-dimensional matrix in which potential helices in the secondary structure appear as diagonal patterns.

Microcomputers↗
PubMedDate not supplied

Comparative characterization on the segmented RNA structure of influenza viruses A/Singapore 1/57 and A/Hong Kong 1/68.

A comparative characterization of the segmented RNA structure of A/Singapore 1/57 and A/Hong Kong 1/68 influenza viruses was done by polyacrylamide gel electrophoresis. The genomes of the viruses studied consist of 6 segment classes, with a total molecular weight of approximately 4 million daltons. The mathematical method suggested allows to define the number of RNA fragments, which is equal to 8-9, with a total molecular weight of about 6 million daltons. Electrophoretic patterns of the viruses investigated have individual features.

Electrophoresis, Polyacrylamide Gel↗
PubMed1975

Murine oncornavirus high-molecular-weight RNA structure thermal stepwise dissociation of 70S murine leukemia-sarcoma virus to subunits and low-molecular-weight associated RNAs.

The thermal dissociation into subunits and low-molecular-weight (LMW) associated RNAs of the aggregate structure of 70S RNA of a murine leukemia sarcoma viral complex was studied. By polyacrylamide-agarose gel electrophoresis, it was found that at low temperature a fraction of the genome was converted into an intermediate population of RNA (Im.P) with an apparent molecular weight of 6.6 times 10-6. At higher temperature, the 70S RNA and the Im.P RNA were successively dissociated into two RNA subunits called "I" and "II" and 70S-associated LMW RNAs. The apparent molecular weight of subunit I was about 5 times 10-6 and that of subunit II was about 3.2 times 10-6. The release of 4S, 5S, 5.5S, and 8S RNAs from 70S RNA at various temperatures was studied by composite polyacrylamide gel electrophoresis. It was found that the nature of hydrogen bonding to the 70S RNA was different for each LMW RNA species. A possible relationship of the association between the subunits and each 70S-associated LMW RNA, based on their T-m values, is discussed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Analysis of formaldehyde-induced Adh mutations in Drosophila by RNA structure mapping and direct sequencing of PCR-amplified genomic DNA.

Two formaldehyde-induced mutations at the Drosophila Adh locus (Adhfn45 and Adhfn46) were analyzed by determining RNA structures at different developmental stages, polymerase chain reaction (PCR) amplification of the affected genomic regions, and direct sequencing of the resulting double-stranded DNA fragments. Adhfn46 adults and larvae accumulate abundant ADH-like distal (adult) and proximal (larval) transcripts that are shorter than transcripts in wild-type flies by a lesion located in the second ADH protein-coding exon. Direct sequencing of the amplified DNA region showed that Adhfn46 contains a 69-bp in-frame deletion that removes 23 amino acids near one border of the second exon. Consistent with these findings, we observed a shorter ADHfn46 protein present at only 3% of wild-type levels. In contrast, Adhfn45 adults and larvae accumulate much smaller amounts of ADH-like distal and proximal transcripts. Both RNAs have an identical aberration in RNA splicing of the 65-base intron sequence. Direct sequencing of the amplified mutated DNA region showed that Adhfn45 contains a 21-bp deletion that removed and rearranged DNA at the 5' splice junction of the 65-bp intron. No ADH cross-reacting material is detected in Adhfn45 flies. Direct-repeat sequences (3-11 bp) are present flanking and within the mutated DNA regions. The patterns of DNA deletion and deletion accompanied by sequence addition at the mutant sites suggest a slipped mispairing mechanism during DNA replication or repair that involves local DNA homology.

Alcohol Dehydrogenase↗
PubMed2025

A small cationic probe for accurate, punctate discovery of RNA tertiary structure.

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

RNA electrostatics↗
PubMed1975

Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qbeta replicase.

Highly purified coliphage Qbeta replicase when incubated without added template synthesizes self-replicating RNA species in an autocatalytic reaction. In this paper we offer strong evidence that this RNA production is directed by templates generated de novo during the lag phase. Contamination of the enzyme by traces of RNA templates was ruled out by the following experimental results: (1) Additional purification steps do not eliminate this RNA production. (2) The lag phase is lengthened to several hours by lowering substrate or enzyme concentration. At a nucleoside triphosphate concentration of 0.15 mM no RNA is produced although the template-directed RNA synthesis works normally. (3) Different enzyme concentrations lead to RNA species of completely different primary structure. (4) Addition of oligonucleotides or preincubation with only three nucleoside triphosphates affects the final RNA sequence. (5) Manipulation of conditions during the lag phase results in the production of RNA structures that are adapted to the particular incubation conditions applied (e.g., RNA resistant to nuclease attack or resistant to inhibitors or even RNAs "addicted to the drug," in the sense that they only replicate in the presence of a drug like acridine orange). RNA species obtained in different experiments under optimal incubation conditions show very similar fingerprint patterns, suggesting the operation of an instruction mechanism. A possible mechanism is discussed.

Acridines↗
PubMed1991

Probing the function of conserved RNA structures in the 30S subunit of Escherichia coli ribosomes.

Ribosomes play an active role in protein biosynthesis. Ribosomal RNA conformation in ribosomal subunits, intramolecular interactions between different rRNA sequences within the confinement of the particles, and intermolecular interactions are presumed necessary to support efficient and accurate protein synthesis. Here we report an analysis of the disposition of 16S rRNA conserved zones centered about positions 525, 1400, and 1500 in 30S subunits. Complementary oligodeoxyribonucleotides in conjunction with nuclease S1 digestion were used to do this. All of the sequences examined in 30S subunits are accessible to DNA probes of 9 to 12 nucleotide residues in length. However, the kinetic characteristics of the respective DNA interactions with 30S particles vary significantly. In addition to the investigation of normal 30S particles, a four base deletion within the 1400 region of 16S rRNA was analyzed. The deletion was made by using synthetic DNAs to target the deletion site for RNase H digestion. The direct in vitro procedure for manipulating rRNA conserves nucleotide modifications. The alteration causes a significant change in the disposition of 16S rRNA in 30S subunits, suggesting a reduction in the freedom of movement of the altered zone in the particle. In a factor-dependent in vitro protein synthesis system primed with MS2 mRNA and altered 30S subunits, there was a 50% decrease in phage coat protein synthesis. The reduction could be due to a decrease in the rate of translation or premature termination of translation. We present evidence here, based on isotopic studies, which supports the latter possibility.

Base Sequence↗
PubMed1990

Silkmoth chorion antisense RNA. Structural characterization, developmental regulation and evolutionary conservation.

Choriogenic follicular cells of the silkmoth Bombyx mori contain significant quantities of antisense RNA transcribed from chorion genes. Antisense RNA derived from a chorion gene with a high content of cysteine, HcB.12, was characterized in detail. The antisense transcripts are initiated downstream from the 3' end of HcB.12 mRNA and extend over 75% of the length of the gene, comprising its entire second exon and part of its intervening sequence. The antisense RNA is devoid of any significant open reading frames and is not polyadenylated. These features, combined with the presence of specific sequence motifs within its transcribed and upstream region, suggest that antisense RNA may be transcribed by RNA polymerase III. Chorion antisense RNA is detectable only in choriogenic follicular cells and appears to be co-ordinately regulated with chorion mRNA. Its cytoplasmic accumulation during choriogenesis parallels that of the corresponding mRNA. Although chorion mRNA is at least five times more abundant than antisense RNA, the latter is present as a single-stranded entity in follicular cytoplasm but can form perfect duplexes with its mRNA complement upon annealing in vitro. The possible involvement of antisense RNA transcription in the pathway that controls the programmed expression of chorion genes at the level of transcription initiation or post-transcriptional processing is discussed.

Animals↗