Studying RNA–Protein Interactions of Pre-mRNA Complexes by Mass Spectrometry

Qamar, Saadia; Kramer, Katharina; Urlaub, Henning

doi:10.1016/bs.mie.2015.02.010

Cited by 6 publications

(11 citation statements)

References 37 publications

(41 reference statements)

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“…Non-cross-linked (oligo)nucleotides were removed by C18 reversed phase chromatography. In a final step, peptide-DNA conjugates were enriched by TiO 2 affinity chromatography 8,27,28 ; Fig. 1b).…”

Section: Resultsmentioning

confidence: 99%

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

et al. 2020

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Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

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Section: Resultsmentioning

confidence: 99%

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

et al. 2020

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“…Site-specific RNP crosslinking e.g. via thiouridine provides unique information about protein-RNA interfaces and entire complex architectures [124,125]. Systematic integration of restraint information from protein-RNA crosslinks is now being used in an automated way [126] and can define and validate RNP models in the future.…”

Section: Biochemical Assaysmentioning

confidence: 99%

“…Dynamic light scattering (DLS) yields information about sample homogeneity and complex size distributions and thus complements SLS data. Low-resolution structural information of protein-RNA interfaces can be obtained from mass spectrometry after crosslinking [124,125]. Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy require the incorporation of pairwise fluorescent or spin labels, respectively, at specific sites.…”

Section: Biophysical Assaysmentioning

confidence: 99%

Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition

Schlundt

Tants

Sattler

2017

Methods

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Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain arrangements and dynamics. Principles of protein-RNA recognition and current approaches are reviewed and illustrated with recent studies.

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“…Along with these advances, several powerful integrated biochemical/bioinformatics approaches can identify both the target RNAs and the specific ribonucleotides recognized by the RNA-binding proteins [41–43]. In contrast, at present, there are no truly high-throughput experimental approaches for identifying interfacial residues in the protein component of a protein–RNA complex, although CLAMP [44] and other cross-linking and combined cross-linking mass spectrometry methods can identify interfacial residues in both the protein and RNA [37, 45, 46]. Despite all of these impressive advances, the cost and effort involved in the experimental determination of protein–RNA complex structures and/or identifying specific RNA-binding residues in proteins, has created a need for reliable computational approaches that can predict the most likely RNA-binding residues in proteins.…”

Section: Introductionmentioning

confidence: 99%

Sequence-Based Prediction of RNA-Binding Residues in Proteins

Walia¹,

EL-Manzalawy

Honavar

et al. 2016

Methods in Molecular Biology

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Identifying individual residues in the interfaces of protein–RNA complexes is important for understanding the molecular determinants of protein–RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein–RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein–RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner.

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Studying RNA–Protein Interactions of Pre-mRNA Complexes by Mass Spectrometry

Cited by 6 publications

References 37 publications

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry

Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition

Sequence-Based Prediction of RNA-Binding Residues in Proteins

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