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At least 55 records · Page 3Linked to original sources
PubMed2002

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles.

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.

Base Sequence↗
PubMed1998

Automatic detection of conserved RNA structure elements in complete RNA virus genomes.

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.

Algorithms↗
PubMed2003

Direct probing of RNA structures and RNA-protein interactions in the HIV-1 packaging signal by chemical modification and electrospray ionization fourier transform mass spectrometry.

RNA hairpins of the HIV-1 packaging signal and their complexes with the nucleocapsid protein p7 (NC) were probed by solvent-accessibility reagents and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The combination of dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMCT) offers the full range of information on base-pairing and solvent exposure concerning the four more abundant ribonucleotides. ESI-FTMS provides a universal method to achieve a direct and unambiguous characterization of alkylated structures, with no need for the different probe-specific procedures required by established methodologies based on gel electrophoresis. It enables us to streamline the optimization of the conditions for probe administration to minimize the incidence of probe-induced distortion of the structures under investigation. Nucleotides located in the single-stranded loops of hairpins SL2, SL3 and SL4 manifested different levels of protection, which were correlated directly to their conformation and structural surroundings. A common feature noted for all the hairpins was the limited susceptibility observed for the guanine base located at the 5'-end of each tetraloop, which assumes a stacked position upon the last base-pair of the double-stranded stems. The remaining loop bases were found to be clearly accessible by modifying reagents in free RNA, but were effectively protected in the NC-hairpin complexes. While this finding is consistent with the proven participation of SL2 and SL3 loops in interactions with NC, it contrasts with prior suggestions that tetraloop bases in SL4 might not be involved directly in NC binding. Alkylation was detected for stem nucleotides, which are not involved in the normal base-pairing and stacking typical of double-stranded structures, such as adenine 15 of the SL2 triple-base platform. Modification of the blunt ends of the double-stranded stems was found to be absent or extremely limited, due to the annealing stabilization introduced by the presence of G-C pairs at the end of the stems structures. Previously undetected alkylation of guanine 3 and guanine 13 in SL4 provides direct evidence of the destabilizing effects induced by the tandem G.U wobbles on the double-stranded structure of this stem, which is thought to be important for the hairpin's biological function.

Base Sequence↗
PubMed2005

Describing RNA structure by libraries of clustered nucleotide doublets.

The rapidly increasing wealth of structural information on RNA and knowledge of its varying roles in biology have facilitated the study of RNA structure using computational methods. Here, we present a new method to describe RNA structure based on nucleotide doublets, where a doublet is any two nucleotides in a structure. We restrict our search to doublets that are close together in space, but not necessarily in sequence, and obtain doublet libraries of various sizes by clustering a large set of doublets taken from a data set of high-resolution RNA structures. We demonstrate that these libraries are able to both capture structural features present in RNA and fit local RNA structure with a high level of accuracy. Libraries ranging in size from ten to 100 doublets are examined, and a detailed analysis shows that a library with as few as 30 doublets is sufficient to capture the most common structural features, while larger libraries would be more appropriate for accurate modeling. We anticipate many uses for these libraries, from annotation to structure refinement and prediction.

Cluster Analysis↗
PubMed1993

RNA structural requirements for RNA binding, replication, and packaging in the yeast double-stranded RNA virus.

Several sites of interaction with viral proteins have been mapped in the plus strand of the Saccharomyces cerevisiae double-stranded RNA virus, ScV. These include a site necessary and sufficient for viral particle binding to plus strands (VBS) and a site necessary and sufficient for interference with replication of viral plus strands (INS). We show that the INS and VBS are identical and that they are necessary and sufficient for packaging. One of the viral RNAs has two adjacent VBS, which have additive INS activity. The second VBS has similar affinity for viral particles as the first but its complex with particles exhibits faster dissociation. Binding to the two sites in the viral RNA is independent but equivalent. This may mean two recognition sites per particle. This is what would be expected for two molecules of the protein thought to be responsible for sequence specific recognition, the cap-pol fusion polypeptide, per particle. Comparison of the secondary structures of several binding sites, as determined by site-directed mutagenesis and by ribonuclease mapping of single- and double-stranded regions, reveals a requirement for a specific loop sequence and two stem regions, in which there is no sequence specificity. Contrary to what was described in previous work, a bulge loop, but not a "bulged" A residue is necessary for binding of the second VBS. A minimal region of 30 bases is enough to cause both packaging and replication.

Base Sequence↗
PubMed2001

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

Adenosine Triphosphate↗
PubMed2005

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA.

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25 degrees C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.

Base Sequence↗
PubMed2001

Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72.

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.

Adenosine Triphosphatases↗
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↗
PubMed1997

How accurately and precisely can RNA structure be determined by NMR?

The great diversity of RNA biological functions has led to widespread interest in RNA structure. Advances in synthetic and spectroscopic techniques have recently allowed the extension of NMR methods of structure determination to RNA structures of significant size and increased biological significance. However, it has not yet been established how accurately and precisely RNA structure can be determined by NMR. The extensive simulations presented here establish credible limits on accuracy and precision of NMR-derived RNA structures and provide quantitative calibrations to evaluate new structures. Synthetic sets of NMR constraints were generated from a crystallographically-derived ribozyme structure. The target structure was then redetermined using approximations and computational protocols derived from our experimental work. The results demonstrate that the structure of RNA molecules of at least 15,000 Da can be determined with precision and accuracy of 1 to 1.5 A, comparable to values obtained for proteins of similar size. Most encouragingly, it is shown that larger, globular and biologically more important RNA structures can be determined with significantly better accuracy and precision than smaller, elongated structures investigated until now.

Base Composition↗
PubMed2005

Nucleotide analogues to investigate RNA structure and function.

RNA plays an essential cellular role in nearly every aspect of the transmission and expression of genetic information, including regulatory roles that have significance for cellular development. Access to RNA bearing synthetic modifications has allowed biological chemists to probe deep into the inner workings of cellular processes. Here, we describe recent advances in harnessing the power of nucleotide analogues to obtain mechanistic and biological insights into RNA structure, function and dynamics.

Catalysis↗
PubMed2000

Database of non-canonical base pairs found in known RNA structures.

Atomic resolution RNA structures are being published at an increasing rate. It is common to find a modest number of non-canonical base pairs in these structures in addition to the usual Watson-Crick pairs. This database summarizes the occurrence of these rare base pairs in accordance with standard nomenclature. The database, http://prion.bchs.uh.edu/, contains information such as sequence context, sugar pucker conformation, anti / syn base conformations, chemical shift, p K (a)values, melting temperature and free energy. Of the 29 anticipated pairs with two or more hydrogen bonds, 20 have been encountered to date. In addition, four unexpected pairs with two hydrogen bonds have been reported bringing the total to 24. Single hydrogen bond versions of five of the expected geometries have been encountered among the single hydrogen bond interactions. In addition, 18 different types of base triplets have been encountered, each of which involves three to six hydrogen bonds. The vast majority of the rare base pairs are antiparallel with the bases in the anti configuration relative to the ribose. The most common are the GU wobble, the Sheared GA pair, the Reverse Hoogsteen pair and the GA imino pair.

Base Pairing↗
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↗
PubMed2003

RNA structure comparison, motif search and discovery using a reduced representation of RNA conformational space.

Given the wealth of new RNA structures and the growing list of RNA functions in biology, it is of great interest to understand the repertoire of RNA folding motifs. The ability to identify new and known motifs within novel RNA structures, to compare tertiary structures with one another and to quantify the characteristics of a given RNA motif are major goals in the field of RNA research; however, there are few systematic ways to address these issues. Using a novel approach for visualizing and mathematically describing macromolecular structures, we have developed a means to quantitatively describe RNA molecules in order to rapidly analyze, compare and explore their features. This approach builds on the alternative eta,theta convention for describing RNA torsion angles and is executed using a new program called PRIMOS. Applying this methodology, we have successfully identified major regions of conformational change in the 50S and 30S ribosomal subunits, we have developed a means to search the database of RNA structures for the prevalence of known motifs and we have classified and identified new motifs. These applications illustrate the powerful capabilities of our new RNA structural convention, and they suggest future adaptations with important implications for bioinformatics and structural genomics.

Algorithms↗