Isoenergetic Microarrays To Study the Structure and Interactions of DsrA and OxyS RNAs in Two- and Three-Component Complexes

et al. 2013

PLoS ONE

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Influenza A virus is a segmented single-stranded (−)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of in vitro chemical mapping and isoenergetic microarray techniques demonstrate that the consensus sequence for this region folds into a hairpin conformation. These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.

Section: Methodsmentioning

confidence: 99%

Secondary Structure of a Conserved Domain in the Intron of Influenza A NS1 mRNA

Priore

et al. 2013

PLoS ONE

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“…Microarray mapping can also be used to study RNA in complex with protein and/or other RNAs ( 77 ). The first models for such studies were non-coding DsrA RNA and OxyS RNA bound to Hfq protein.…”

Section: Developing Rna-like and Isoenergetic Microarrays For Probingmentioning

confidence: 99%

“…The bar graph demonstrates intensity of binding to probes (at selected positions) of DsrA RNA in the absence and presence of Hfq. Various patterns hybridization of DsrA RNA is related with different positioning probes ( 77 ).…”

Section: Developing Rna-like and Isoenergetic Microarrays For Probingmentioning

confidence: 99%

Microarrays for identifying binding sites and probing structure of RNAs

Turner

2014

Nucleic Acids Research

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154

124

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.

“…Moreover, sRNA binding to wild type and mutated Hfq 29,41 RybB, 43 OxyS, 42 IstR1, 38 ChiX, 40 RybB. Linear fitting of the data for IstR1 and SgrS provided k off values of 1.8 × 10 −4 s −1 and 4.5 × 10 −4 s −1 , respectively; (C) equilibrium assay monitoring competition of unlabeled sRNAs against 32 P-DsrA bound to Hfq.…”

mentioning

confidence: 99%

Structure of Bacterial Regulatory RNAs Determines Their Performance in Competition for the Chaperone Protein Hfq

et al. 2015

Bacterial regulatory RNAs require the chaperone protein Hfq to enable their pairing to mRNAs. Recent data showed that there is a hierarchy among sRNAs in the competition for access to Hfq, which could be important for the tuning of sRNA-dependent translation regulation. Here, seven structurally different sRNAs were compared using filter-based competition assays. Moreover, chimeric sRNA constructs were designed to identify structure elements important for competition performance. The data showed that besides the 3'-terminal oligouridine sequences also the 5'-terminal structure elements of sRNAs were essential for their competition performance. When the binding of sRNAs to Hfq mutants was compared, the data showed the important role of the proximal and rim sites of Hfq for the binding of six out of seven sRNAs. However, ChiX sRNA, which was the most efficient competitor, bound Hfq in a unique way using the opposite-distal and proximal-faces of this ring-shaped protein. The data indicated that the simultaneous binding to the opposite faces of Hfq was enabled by separate adenosine-rich and uridine-rich sequences in the long, single-stranded region of ChiX. Overall, the results suggest that the individual structural composition of sRNAs serves to tune their performance to different levels resulting in a hierarchy of sRNAs in the competition for access to the Hfq protein.