Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry

Kühn-Hölsken, Eva; Dybkov, Olexandr; Sander, Björn; Lührmann, Reinhard; Urlaub, Henning

doi:10.1093/nar/gkm540

Cited by 21 publications

(37 citation statements)

References 46 publications

(72 reference statements)

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“…Therefore, RNA hydrolysis using nucleases generating mono-to tetra-nucleotides (e.g. a combination of RNase A and T1 or benzonase) is desirable, especially for ESI-MSMS analysis of peptide-nucleotide crosslinks [78][79][80]. Accordingly, Steen et al used hydrolysis with phosphodiesterase I, followed by alkaline phosphatase to remove phosphate groups, on a peptide-DNA conjugate [80], whereas Geyer et al used hydrofluoric acid to hydrolyse the crosslinked RNA moiety [78].…”

Section: Purification Strategiesmentioning

confidence: 99%

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Schmidt

Kramer

Urlaub

2012

Journal of Proteomics

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Section: Purification Strategiesmentioning

confidence: 99%

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Schmidt

Kramer

Urlaub

2012

Journal of Proteomics

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“…Moreover, formaldehyde and other chemical cross-linkers may not enter the cores of large complexes. This problem can be avoided by cross-linking proteins and RNA with UV light, and several techniques have been reported (3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

“…These approaches can detect cross-links on both protein and RNA but primer extension mapping on long RNAs is not practical without prior knowledge of the approximate cross-linking site and MS analyses require up to 50 pmol of RNP (5). The cross-linking and immunoprecipitation (CLIP) method identified protein-RNA interaction sites in mammalian cells by cloning of the covalentlyattached RNAs (6,7).…”

mentioning

confidence: 99%

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Granneman

Kudla

Petfalski

et al. 2009

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

328

446

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The U3 small nucleolar ribonucleoprotein (snoRNP) plays an essential role in ribosome biogenesis but, like many RNA-protein complexes, its architecture is poorly understood. To address this problem, binding sites for the snoRNP proteins Nop1, Nop56, Nop58, and Rrp9 were mapped by UV cross-linking and analysis of cDNAs. Cross-linked protein-RNA complexes were purified under highly-denaturing conditions, ensuring that only direct interactions were detected. Recovered RNA fragments were amplified after linker ligation and cDNA synthesis. Cross-linking was successfully performed either in vitro on purified complexes or in vivo in living cells. Cross-linking sites were precisely mapped either by Sanger sequencing of multiple cloned fragments or direct, high-throughput Solexa sequencing. Analysis of RNAs associated with the snoRNP proteins revealed remarkably high signal-to-noise ratios and identified specific binding sites for each of these proteins on the U3 RNA. The results were consistent with previous data, demonstrating the reliability of the method, but also provided insights into the architecture of the U3 snoRNP. The snoRNP proteins were also cross-linked to pre-rRNA fragments, with preferential association at known sites of box C/D snoRNA function. This finding demonstrates that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment. The techniques reported here should be widely applicable to analyses of RNA-protein interactions.ribosome synthesis ͉ RNA modification ͉ RNA processing ͉ RNP structure ͉ yeast P roteomic approaches have identified many factors involved in ribosome synthesis in yeast, but we still lack detailed understanding of the architecture of the preribosomes and small nucleolar ribonucleoprotein (snoRNP) complexes that are required for their maturation. Several methods have been described that allow identification of protein-RNA interaction sites in native particles. RNA immunoprecipitation uses formaldehyde to cross-link RNA to proteins (1, 2). Caveats of this method are that formaldehyde also cross-links proteins to proteins and the immunoprecipitation step is performed under semidenaturing conditions, so a positive result does not demonstrate direct RNA-protein interaction, and the spatial resolution of the technique is low. Moreover, formaldehyde and other chemical cross-linkers may not enter the cores of large complexes. This problem can be avoided by cross-linking proteins and RNA with UV light, and several techniques have been reported (3-7).UV-induced protein-RNA cross-links can be detected by primer extension analysis on the RNA and by MALDI-MS on the protein (4). These approaches can detect cross-links on both protein and RNA but primer extension mapping on long RNAs is not practical without prior knowledge of the approximate cross-linking site and MS analyses require up to 50 pmol of RNP (5). The cross-linking and immunoprecipitation (CLIP) method identified protein-RNA interaction sites in mammalian cells by cloning of the covalentlya...

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“…The cross-linked nucleotide is determined by calculating the mass difference between the entire mass of the cross-linked species and the mass of the cross-linked peptide. In addition, marker ions of the cross-linked nucleotides in the lower [37, 43,44]. Currently, electrospray ionization (ESI) MS coupled to a nano-liquid chromatography (LC) is the method of choice for analyzing such cross-links.…”

Section: Mass Spectrometry Analysismentioning

confidence: 99%

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

Sharma

Hrle

Kramer

et al. 2015

Methods

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a b s t r a c tRibonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different singleand multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 -RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

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Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry

Cited by 21 publications

References 46 publications

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

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