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

Schmidt, Carla; Kramer, Katharina; Urlaub, Henning

doi:10.1016/j.jprot.2012.04.030

Cited by 49 publications

(77 citation statements)

References 118 publications

(196 reference statements)

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“…Importantly, subsequent tryptic digestion of the RNA-bound LysC/ArgC fragments yields two classes of peptides: the portion that still remains crosslinked to the RNA (X-link) and its neighboring peptides (N-link) (Figure 1A). While the directly crosslinked peptides (X-link) are difficult to identify due to the heterogeneous mass shift induced by the residual nucleotides (Kramer et al., 2014, Schmidt et al., 2012), the native peptides adjacent to the crosslinking site (N-link) can be identified by standard MS and peptide search algorithms. The original RNA-bound region of the RBP (i.e., RBDpep; Figure 1A), which includes both the crosslinked peptide (X-link) and its unmodified neighboring peptides (N-link), is then re-derived in silico by extending the MS-identified peptides to the two nearest LysC or ArgC cleavage sites.…”

Section: Resultsmentioning

confidence: 99%

“…To identify the interaction sites of such proteins with RNA, UV crosslinking followed by extensive RNase treatment has been used to detect the peptide mass shift induced by the crosslinked RNA remnant via mass spectrometry (Schmidt et al., 2012). While conceptually simple, the mass heterogeneity of the nucleotide remnant has rendered this approach challenging in practice.…”

Section: Introductionmentioning

confidence: 99%

“…While conceptually simple, the mass heterogeneity of the nucleotide remnant has rendered this approach challenging in practice. Some RBDs have been characterized in vitro using this approach (reviewed in Schmidt et al., 2012), and a sophisticated algorithm allowed assignment of 257 binding sites from 124 proteins in yeast (Kramer et al., 2014). While informative, this data set is strongly enriched for interactions mediated by RRMs, because the challenging identification of peptides with aberrant mass spectra requires both abundance and high crosslinking efficiency for detection.…”

Section: Introductionmentioning

confidence: 99%

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Comprehensive Identification of RNA-Binding Domains in Human Cells

et al. 2016

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SummaryMammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comprehensive Identification of RNA-Binding Domains in Human Cells

et al. 2016

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“…Overall, although the debate whether native protein structure is maintained in the gas phase continues, the future is bright for structural MS analysis of proteins and their complexes, as illustrated by a growing body of work of, for instance, protein–lipid [26, 74, 75], protein–RNA [76], protein–DNA [77], protein–drug [78], and protein–protein complexes [79, 80]. …”

Section: Protein Structure In the Gas Phasementioning

confidence: 99%

Native Mass Spectrometry: What is in the Name?

Leney

Heck

2016

J. Am. Soc. Mass Spectrom.

516

480

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Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ

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“…during different stages of the RNA cycle, during cell cycle progression, and in cells that have been treated with different extracellular stimuli (stress conditions). Moreover, because many RNA-binding proteins interact either weakly or transiently with their target RNA throughout its ‘life’ cycle, it is crucial to streamline existing methods or to develop novel more accurate, high-throughput technologies that exploit crosslinking-MS (206,207) and next-generation sequencing (18,121,208). Thus far, among the major caveats of RNA-centric methods is their inability to explore RNA-binding proteins that associate with low abundance transcripts inside living cells ( in vivo purification strategies).…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

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

Cited by 49 publications

References 118 publications

Comprehensive Identification of RNA-Binding Domains in Human Cells

Comprehensive Identification of RNA-Binding Domains in Human Cells

Native Mass Spectrometry: What is in the Name?

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

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