Fast and unbiased purification of RNA-protein complexes after UV cross-linking

Urdaneta, Erika C.; Beckmann, Benedikt M.

doi:10.1016/j.ymeth.2019.09.013

Cited by 33 publications

(37 citation statements)

References 44 publications

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“…Overcoming these could result in major advances in the field and a better characterization of multiple RNPs, even the ones difficult to study, such as those associated with low-copy-number RNAs. With the recent publication of organic phase separation protocols such as Cross-Linked RNA eXtraction (XRNAX) [ 21 ], orthogonal organic phase separation (OOPS) [ 22 ] and Phenol-Toluol extraction (PTex) [ 23 ], we address these challenges and propose future experimental strategies that build on these methods.…”

Section: Opening Doors To the Rna-binding Proteomementioning

confidence: 99%

“…Since cross-linking an RNP is based on the physical proximity of the RNA and the protein rather than on the physiological role of the protein, the term RBP becomes broader or a new term has to be introduced, as suggested by Urdaneta et al Because of this proximity binding, the question of whether the identified binding proteins are functional binders or just spatially close to a given RNA molecule during the time of cross-linking can arise. Together with the fact that a lot is still unknown about the role and importance of numerous transient RNA–protein interactions, it is advisable to experimentally validate identified targets, no matter how stringent the purification procedure [ 23 , 24 ].…”

Section: Opening Doors To the Rna-binding Proteomementioning

confidence: 99%

“…Chemical cross-linking could be used for tissues where UV light cannot penetrate the whole tissue. However, the recently noticed property of frozen powdered tissue, which can be used as source material for UV cross-linking, could circumvent this problem [ 23 ]. In summary, chemical cross-linking and UV cross-linking are complementary techniques and can be useful RNA-centric methods to use as a first identification step, followed up by protein-centric approaches to verify whether the protein has the expected RNA binding property.…”

Section: Cross-linking Proteins To Rna or Not And How?mentioning

confidence: 99%

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Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

2020

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The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

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Section: Opening Doors To the Rna-binding Proteomementioning

confidence: 99%

Section: Opening Doors To the Rna-binding Proteomementioning

confidence: 99%

Section: Cross-linking Proteins To Rna or Not And How?mentioning

confidence: 99%

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Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

2020

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“…Diese neue Methode, PTex (Phenol Toluol extraction, Abb. 1), ermöglicht es daher, alle RNPs aus diversen Probenmaterialien ohne Antikörper oder komplementäre Nukleinsäuresequenzen von anderen ungebundenen RNAs oder Proteinen zu trennen und anzureichern [5,6]. Die einzigen Voraussetzungen für PTex bestehen darin, dass das Ausgangsmaterial mit UV-Licht effi zient bestrahlt wird und danach ohne Zugabe von denaturierenden Reagenzien lysiert werden kann.…”

Section: Ptex: Flüssig-flüssig-extraktion Reichert Rna-protein-kompleunclassified

Systemweite Anreicherung von RNA-Protein-Komplexen

Pfister

Beckmann

2020

Biospektrum

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RNA-protein complexes (RNPs) are key players in cell physiology, especially in the context of RNA metabolism. However, their analysis has been dependent on specific protein epitopes or RNA sequence elements, preventing unbiased and cell-wide studies. We developed a three-step protocol, termed Phenol Toluol extraction (PTex), to isolate the complete suite of RNPs in cells, solely based on their unique physicochemical properties after UV-cross-linking. PTex, along with other recently developed unbiased techniques, has the potential to guide the way to cell-wide analysis of RNA-protein interactions.

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“…Since UV-crosslinking has very low efficiency, antisense probe-based purification methods usually require a large number of cells (∼100-800 million) (Lin et al, 2019; McHugh et al, 2015), which may not be feasible for slow-growing model systems such as primary cell cultures. Moreover, UV-crosslinking can induce RNA alterations like modifications (Wurtmann and Wolin, 2009) that could change binding affinity of RNA to certain RBPs (Bernard et al, 2012) and impair downstream protein analysis (Urdaneta and Beckmann, 2019). An alternative approach, tagging of endogenous RNA requires genetic manipulation and may interfere with endogenous RNA functions (Laprade et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

In vivodiscovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

Lin

Fonseca

Corona

et al. 2020

Preprint

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KL). 10 11 2 Functional and mechanistic annotation of uncharacterized long noncoding RNAs is 12 challenging and often requires identification of interacting proteins. Here we report 13 RiboPro, a flexible method that leverages the RNA-binding specificity of inactive 14 Cas13b and proximity labeling activity of peroxidase APEX to permit rapid and unbiased 15 discovery of target RNA binding proteins in vivo. RiboPro of poly(A)+ RNA reveals 16 insights into poly (A)+ RNA nucleocytoplasmic transport, localization, turnover, and 17 higher-order structure. 18 19LncRNA transcripts do not encode protein, but they nonetheless play myriad roles in 20 physiology and disease 1 . The spectrum of RNA binding proteins (RBPs) associated with a 21 lncRNA transcript will largely dictate lncRNA activities 2 . RBPs control critical RNA activities 22

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Fast and unbiased purification of RNA-protein complexes after UV cross-linking

Cited by 33 publications

References 44 publications

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

Systemweite Anreicherung von RNA-Protein-Komplexen

In vivodiscovery of RNA proximal proteins in human cells via proximity-dependent biotinylation

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