Simultaneous profiling of the RNA targets of two RNA-binding proteins using TRIBE-STAMP.
| Autor: | Flamand MN; Centre de Recherche du CHU de Québec-Université Laval, Axe Neurosciences, Québec, QC, Canada; Département de Psychiatrie et de Neurosciences, Faculté de Médecine, Université Laval, Québec, QC, Canada. Electronic address: mathieu.flamand@crchudequebec.ulaval.ca., Meyer KD; Department of Biochemistry, Duke University School of Medicine, Durham, NC, United States; Department of Neurobiology, Duke University School of Medicine, Durham, NC, United States; Department of Cell Biology, Duke University School of Medicine, Durham, NC, United States. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in enzymology [Methods Enzymol] 2024; Vol. 705, pp. 127-157. Date of Electronic Publication: 2024 Aug 17. |
| DOI: | 10.1016/bs.mie.2024.07.008 |
| Abstrakt: | RNA-binding proteins (RBPs) are central players in RNA homeostasis and the control of gene expression. The identification of RBP targets, interactions, and the regulatory networks they control is crucial for understanding their cellular functions. Traditional methods for identifying RBP targets across the transcriptome have been insightful but are limited by their focus on a single RBP at a time and their general inability to identify individual RNA molecules that are bound by RBPs of interest. Recently, we overcame these limitations by developing TRIBE-STAMP, a method which enables concurrent identification of the RNA targets of two RBPs of interest with single-molecule resolution. TRIBE-STAMP works by tagging desired RBPs with either the ADAR or APOBEC1 RNA editing enzymes and expressing them in cells, followed by RNA-seq. Subsequent computational identification of A-to-I and C-to-U editing events enables the simultaneous identification of the ADAR- and APOBEC1-fused RBP target RNAs, respectively. Here, we present a detailed protocol for TRIBE-STAMP, including considerations for fusion protein expression in cells and step-by-step computational analysis of sequencing data. TRIBE-STAMP is a simple and highly versatile approach for single-molecule identification of the targets of RBPs which enables unprecedented insights into the biological interplay between RBP pairs in cells. (Copyright © 2024. Published by Elsevier Inc.) |
| Databáze: | MEDLINE |
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