A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

Matia-González, Ana M.; Iadevaia, Valentina; Gerber, André P.

doi:10.1016/j.ymeth.2016.10.005

Cited by 40 publications

(38 citation statements)

References 37 publications

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“…A protocol suitable for the isolation of endogenous mRNA, called TRIP (tandem RNA isolation procedure), has also been described (Matia-González et al 2017). TRIP uses short (21-24 nt) 3 ′ -biotinylated 2 ′ -O-methylated antisense RNA oligonucleotides.…”

Section: Discussionmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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Section: Discussionmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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“…In the end, the functions of 3 ′ UTRs are determined by the bound RBPs and their associated effector proteins. Whereas cross-linking immunoprecipitation (CLIP) identifies all mRNAs bound by a single RBP, complementary approaches detect all RBPs that bind single 3 ′ UTRs (Chu et al 2015;Matia-Gonzalez et al 2017). However, the identification of functionally relevant RBP-binding sites is complicated as the motifs are often degenerate.…”

Section: Identification Of Rna-binding Proteinsmentioning

confidence: 99%

What Are 3′ UTRs Doing?

Mayr

2018

Cold Spring Harb Perspect Biol

386

278

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SUMMARY3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation. In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins. This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations. Therefore, 3' UTR-mediated information transfer can regulate protein features that are not encoded in the amino acid sequence. This review summarizes both 3' UTR functions-the regulation of mRNA and protein-based processes-and highlights how each 3' UTR function was discovered with a focus on experimental approaches used and the concepts that were learned. This review also discusses novel approaches to study 3' UTR functions in the future by taking advantage of recent advances in technology.

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“…However, so far, most studies for de-novo identification of transcript-bound proteins have focused on abundant non-coding or exogenously expressed RNAs and were only applied in cell culture [11][12][13][14][15][16] . Recently, a tandem purification approach for isolation of mRNA-protein complexes from yeast, C. elegans, and human cells has been proposed, but transcript enrichment has been reported to be limited 47 . Here, we present vIPR, a highly specific and sensitive method for de-novo identification of proteins interacting with an mRNA of interest in vivo in C. elegans.…”

Section: Discussionmentioning

confidence: 99%

Identification of proteins and miRNAs that specifically bind an mRNA in vivo

2019

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Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germlinespecific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

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A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

Cited by 40 publications

References 37 publications

Specific RNP capture with antisense LNA/DNA mixmers

Specific RNP capture with antisense LNA/DNA mixmers

What Are 3′ UTRs Doing?

Identification of proteins and miRNAs that specifically bind an mRNA in vivo

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