Cross-Linking and Immunoprecipitation of Nuclear RNA-Binding Proteins

Li, Quan; Uemura, Yuri; Kawahara, Yukio

doi:10.1007/978-1-4939-2253-6_15

Cited by 10 publications

(8 citation statements)

References 15 publications

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“…PAR-CLIP analysis was carried out as described previously (Hafner et al 2010;Lebedeva et al 2011;Yokoshi et al 2014;Li et al 2015) with some modifications. Briefly, SH-SY5Y cells were cultured on 60 15-cm culture dishes in the presence of 100 lM 4-SU (Sigma, St Louis, MO, USA) for 14-16 h. After the medium had been aspirated, the cells were crosslinked on ice by irradiation with 365 nm UV at 150 mJ/cm 2 in a UV cross-linker (FS-1500L; Funakoshi).…”

Section: Par-clip Analysismentioning

confidence: 99%

“…; Li et al . ). Recently, we have successfully elucidated the function of Ataxin‐2, which promotes mRNA stability by direct binding to target mRNAs, through the comprehensive identification of its targets using PAR‐CLIP (Yokoshi et al .…”

Section: Introductionmentioning

confidence: 97%

“…This results in strong cross-linking between the 4-SU in the RNA and bound RBPs. In addition, cross-linked 4-SU is converted into cytosine (C) rather than thymidine (T) during reverse transcription, which thereby marks the binding site of the protein at a single-nucleotide resolution (Hafner et al 2010;Lebedeva et al 2011;Yokoshi et al 2014;Li et al 2015). Recently, we have successfully elucidated the function of Ataxin-2, which promotes mRNA stability by direct binding to target mRNAs, through the comprehensive identification of its targets using PAR-CLIP (Yokoshi et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

et al. 2017

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Matrin3 is an RNA-binding protein that is localized in the nuclear matrix. Although various roles in RNA metabolism have been reported for Matrin3, in vivo target RNAs to which Matrin3 binds directly have not been investigated comprehensively so far. Here, we show that Matrin3 binds predominantly to intronic regions of pre-mRNAs. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) analysis using human neuronal cells showed that Matrin3 recognized pyrimidine-rich sequences as binding motifs, including the polypyrimidine tract, a splicing regulatory element. Splicing-sensitive microarray analysis showed that depletion of Matrin3 preferentially increased the inclusion of cassette exons that were adjacent to introns that contained Matrin3-binding sites. We further found that although most of the genes targeted by polypyrimidine tract binding protein 1 (PTBP1) were also bound by Matrin3, Matrin3 could control alternative splicing in a PTBP1-independent manner, at least in part. These findings suggest that Matrin3 is a splicing regulator that targets intronic pyrimidine-rich sequences.

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Section: Par-clip Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

et al. 2017

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“…RIP and CLIP assays combined with microarrays or RNA-seq allow genome-wide analysis of RBP-RNA interactions (Jain et al, 2011; König et al, 2011; Li et al, 2015; Milek et al, 2012; Modic et al, 2013; Re et al, 2014). RIP analysis on the lens epithelial cell line 21EM15 identified Hspb1 ( Hsp27 ) among Tdrd7-RNA targets (Lachke et al, 2011).…”

Section: Protein-nucleic Acid Interaction-profiling For Lens Grnsmentioning

confidence: 99%

Systems biology of lens development: A paradigm for disease gene discovery in the eye

Anand

Lachke

2017

Experimental Eye Research

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Over the past several decades, the biology of the developing lens has been investigated using molecular genetics-based approaches in various vertebrate model systems. These efforts, involving target gene knockouts or knockdowns, have led to major advances in our understanding of lens morphogenesis and the pathological basis of cataracts, as well as of other lens related eye defects. In particular, we now have a functional understanding of regulators such as Pax6, Six3, Sox2, Oct1 (Pou2f1), Meis1, Pnox1, Zeb2 (Sip1), Mab21l1, Foxe3, Tfap2a (Ap2-alpha), Pitx3, Sox11, Prox1, Sox1, c-Maf, Mafg, Mafk, Hsf4, Fgfrs, Bmp7, and Tdrd7 in this tissue. However, whether these individual regulators interact or their targets overlap, and the significance of such interactions during lens morphogenesis, is not well defined. The arrival of high-throughput approaches for gene expression profiling (microarrays, RNA-sequencing (RNA-seq), etc.), which can be coupled with chromatin immunoprecipitation (ChIP) or RNA immunoprecipitation (RIP) assays, along with improved computational resources and publically available datasets (e.g. those containing comprehensive protein-protein, protein-DNA information), presents new opportunities to advance our understanding of the lens tissue on a global systems level. Such systems-level knowledge will lead to the derivation of the underlying lens gene regulatory network (GRN), defined as a circuit map of the regulator-target interactions functional in lens development, which can be applied to expedite cataract gene discovery. In this review, we cover the various systems-level approaches such as microarrays, RNA-seq, and ChIP that are already being applied to lens studies and discuss strategies for assembling and interpreting these vast amounts of high-throughput information for effective dispersion to the scientific community. In particular, we discuss strategies for effective interpretation of this new information in the context of the rich knowledge obtained through the application of traditional single-gene focused experiments on the lens. Finally, we discuss our vision for integrating these diverse high-throughput datasets in a single web-based user-friendly tool iSyTE (integrated Systems Tool for Eye gene discovery) – a resource that is already proving effective in the identification and characterization of genes linked to lens development and cataract. We anticipate that application of a similar approach to other ocular tissues such as the retina and the cornea, and even other organ systems, will significantly impact disease gene discovery.

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“…A similar approach but followed by microarray, was used by Khalil and colleagues to show that about 20% of lincRNAs associates with PRC2 complex (Khalil et al, 2009). The main limitations of RIP approaches are (i) the possible loss/gain of targets during the extraction, including non-specific interactions that may form after cell lysis (McHugh et al, 2014), (ii) the identification and exclusion of false positive hits, and (iii) the determination of the exact location of the binding site of RBPs that will require subsequent motif analysis (Li et al, 2015). To overcome these drawbacks, crosslinking of the RNA-protein complexes in the living cells was introduced.…”

Section: Protein-centric Methodsmentioning

confidence: 99%

Probing Long Non-coding RNA-Protein Interactions

Barra

Leucci

2017

Front. Mol. Biosci.

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Non-coding RNA sequences outnumber the protein-coding genes in the human genome, however our knowledge of their functions is still limited. RNA-binding proteins follow the transcripts, including non-coding RNAs, throughout their life, regulating not only maturation, nuclear export, stability and eventually translation, but also RNA functions. Therefore, development of sophisticated methods to study RNA-protein interactions are key to the systematic characterization of lncRNAs. Although mostly applicable to RNA-protein interactions in general, many approaches, especially the computational ones, need adjustment to be adapted to the length and complexity of lncRNA transcripts. Here we critically review all the wet lab and computational methods to study lncRNA-protein interactions and their potential to clarify the dark side of the genome.

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Cross-Linking and Immunoprecipitation of Nuclear RNA-Binding Proteins

Cited by 10 publications

References 15 publications

Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

Systems biology of lens development: A paradigm for disease gene discovery in the eye

Probing Long Non-coding RNA-Protein Interactions

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