Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells

Damme, Philip Van; Li, Qing; Zhang, Bo; Bai,; Lee, Myeong Soo; Yesselman,; J, Lu; Velema,

doi:10.1101/2021.11.19.469208

Cited by 2 publications

(3 citation statements)

References 61 publications

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“…4A ) [34] suggesting an interaction between RBM7 and the 7SK RNP even in the absence of DNA damage. Although the 7SK SL3 secondary structure and boundaries are not well characterized, independent chemical mapping studies report SL3 boundaries between residues 200-274 [81-83]. Crosslinking was enriched in the upper stem of 7SK SL3 and observed to be highest for residues A245-U246, with additional crosslinking observed adjacent to this site as well as G232-C235 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

Sobeh

Eichhorn

2022

Preprint

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The 7SK ribonucleoprotein (RNP) is a critical regulator of eukaryotic transcription. Recently, RNA binding motif 7 (RBM7), which contains an RNA recognition motif (RRM), was reported to associate with 7SK RNA and core 7SK RNP protein components in response to DNA damage. However, little is known about the mode of RBM7-7SK RNA recognition. Here, we recombinantly expressed and purified RBM7 RRM constructs and found that constructs containing extended C-termini have increased solubility and stability compared to shorter constructs. To identify potential RBM7-7SK RNA binding sites, we analyzed deposited data from in cellulo crosslinking experiments and found that RBM7 crosslinks specifically to the distal region of 7SK stem-loop 3 (SL3). Electrophoretic mobility shift assays and NMR chemical shift perturbation experiments showed weak binding to 7SK SL3 constructs in vitro. Together, these results provide new insights into RBM7 RRM folding and recognition of 7SK RNA.Graphical abstractHighlightsExtending the RBM7 RRM C-terminus improves protein expression and solubilityThe RBM7 RRM interacts weakly with 7SK stem-loop 3 single-stranded RNASolution state NMR dynamics measurements support presence of additional β4’ strand

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Section: Resultsmentioning

confidence: 99%

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

Sobeh

Eichhorn

2022

Preprint

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“…Chemical probes capable of directly capturing through-space interactions are a very exciting recent development in HTS-based structure probing. By extending SHAPE chemistry, bifunctional acylation reagents have been generated that capture two 2′-hydroxyl residues that are in close spatial proximity [42][43][44] (Fig. 1c).…”

Section: Mapping Rna-rna Base-pairing and Through-space Interactionsmentioning

confidence: 99%

“…1c). For example, spatial 2′-hydroxyl acylation reversible cross-linking (SHARC) uses bifunctional acylation reagents with flexible linkers to cross-link spatially proximal nucleotides 43 . In this case, the linker length is assumed to set the cross-linking distance and, hence, the structural distance between two sites of SHAPE adduct formation.…”

Section: Mapping Rna-rna Base-pairing and Through-space Interactionsmentioning

confidence: 99%

Probing the dynamic RNA structurome and its functions

2022

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RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome -the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.Sections glyoxal and other α-ketoaldehydes, which react with unpaired guanines 14 , and carbodiimide reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which display selectivity for unpaired guanines and uracils, or for G:U wobble pairs 15,16 .By contrast, chemical probing by selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) measures the flexibility of the RNA backbone 17 , which is generally used as a proxy for base-pairing: when the SHAPE reagent forms an adduct with the 2′-OH of a structurally flexible nucleotide, the position is assumed to be unpaired. Moreover, specific structural states have been shown to promote SHAPE Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author selfarchiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

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Chemical Reversible Crosslinking Enables Measurement of RNA 3D Distances and Alternative Conformations in Cells

Cited by 2 publications

References 61 publications

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

Probing the dynamic RNA structurome and its functions

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