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UvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.

ABSTRACT: RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation, and stability of mRNAs. We describe ultraviolet crosslinking and affinity purification (uvCLAP), an easy-to-use, robust, reproducible, and high-throughput method to determine in vivo targets of RBPs. uvCLAP is fast and does not rely on radioactive labeling of RNA. We investigate binding of 15 RBPs from fly, mouse, and human cells to test the method's performance and applicability. Multiplexing of signal and control libraries enables straightforward comparison of samples. Experiments for most proteins achieve high enrichment of signal over background. A point mutation and a natural splice isoform that change the RBP subcellular localization dramatically alter target selection without changing the targeted RNA motif, showing that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.

SUBMITTER: Maticzka D

PROVIDER: S-EPMC5861125 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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uvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.

Maticzka Daniel D Ilik Ibrahim Avsar IA Aktas Tugce T Backofen Rolf R Akhtar Asifa A

Nature communications 20180320 1

RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation, and stability of mRNAs. We describe ultraviolet crosslinking and affinity purification (uvCLAP), an easy-to-use, robust, reproducible, and high-throughput method to determine in vivo targets of RBPs. uvCLAP is fast and does not rely on radioactive labeling of RNA. We investigate binding of 15 RBPs from fly, mouse, and human cells to test the method's perfor ...[more]

PMID: 29559621

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Project description:Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila, the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFβ/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference-based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143-53. ©2017 AACR.

Dataset Information

UvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.

Publications

uvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.

Similar Datasets

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