The NMR and X-ray Structures of the Saccharomyces cerevisiae Vts1 SAM Domain Define a Surface for the Recognition of RNA Hairpins

Aviv, Tzvi; Amborski, Andrew N.; Zhao, Xin; Kwan, Janice; Johnson, Phyllis E.; Sicheri, Frank; Donaldson, Logan W.

doi:10.1016/j.jmb.2005.11.066

Cited by 20 publications

(22 citation statements)

References 28 publications

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“…These apparent affinities were comparable to known Vts1 target elements that we doped into our library (0.1%) as a positive control for RNA folding and protein binding. They also were concordant with published bulk solution measurements (21,22,27) (Fig. 1F; see Materials and Methods for further discussion).…”

Section: Resultssupporting

confidence: 90%

“…S4 A and B). Our data thus reiterate yet significantly expand the consensus CNGGN 0-3 hairpin loop defined by decades of targeted biochemical studies in a wide range of organisms (20)(21)(22) (Fig. 2A).…”

Section: Resultssupporting

confidence: 74%

“…S7B). Indeed, Lys467 mutant proteins exhibit reduced substrate binding (21,22). Among all Vts1 targets, our data revealed that among endogenous targets C:G base pairing between loop positions 1 and 4 is preferred (∼87% of targets) and correlates with the strongest apparent affinities (Fig.…”

Section: Resultsmentioning

confidence: 67%

“…3 A and B). Although no stem composition preferences have previously been reported and no direct stem-Vts1 contacts are observable in the available structures (21,27), TGA allowed us to observe approximately 10-fold stronger binding under these conditions to one of these variants (SRE3) (Fig. S6).…”

Section: Resultsmentioning

confidence: 74%

“…We applied our method to characterize the interactions of the conserved RNA binding domain of a sequence-and structure-specific RBP (Vts1 in Saccharomyces cerevisiae; Smaug in metazoans). We chose to study Vts1 because of its biological significance as a key regulator of RNA stability in development (20) and because decades of prior study provided a gold standard against which to benchmark our results (21)(22)(23)(24)(25)(26)(27).…”

mentioning

confidence: 99%

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Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

She

Chakravarty

Layton

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

108

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RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptomewide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.RNA | next-generation sequencing | systems biochemistry | RNA binding proteins | Vts1 R NA-binding proteins (RBPs) constitute 5-10% of the eukaryotic proteome (1-3) and collectively govern the localization, translation, and decay of virtually every transcript (4-6). Despite the ubiquity of RBPs and their central importance in gene regulation, decoding the links between RNA primary sequence and its cadre of regulators remains a major unresolved challenge (7). Current approaches for characterizing RBP function generally involve trade-offs between throughput, comprehensiveness, and quantitative precision. Biophysical measurements can be made with targeted biochemical approaches such as electrophoretic mobility shift assays (EMSAs) or fluorescence polarization (FP) (8, 9), but these methods can only interrogate known RNA-protein interactions and are inherently lowthroughput. Selection-based approaches [e.g., in vitro selection, high-throughput sequencing of RNA, and sequence-specificity landscapes (SEQRS)/RNA bind-n-seq (RBNS)] achieve higher throughput, but these techniques remove binding sites from their natural sequence context and identify "winners" based on more than simple affinity (10). Transcriptome-wide methods, which often use cross-linking and immunoprecipi...

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 74%

mentioning

confidence: 99%

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Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

She

Chakravarty

Layton

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

108

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Amodiaquine Nonspecifically Binds Double Stranded and Three‐Way Junction DNA Structures

Slavkovic,

Shoara,

Kaiyum

et al. 2024

ChemBioChem

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The 4‐aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine‐binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of mM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three‐way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson‐Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy.These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

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Current awareness on yeast

2006

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals ‐ search completed 17th. May 2006)

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The NMR and X-ray Structures of the Saccharomyces cerevisiae Vts1 SAM Domain Define a Surface for the Recognition of RNA Hairpins

Cited by 20 publications

References 28 publications

Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

Amodiaquine Nonspecifically Binds Double Stranded and Three‐Way Junction DNA Structures

Current awareness on yeast

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