Simultaneous profiling of the RNA targets of two RNA-binding proteins using TRIBE-STAMP

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• RNA-binding proteins (RBPs) are critical for RNA homeostasis and gene expression regulation.
• Identifying RBP targets and their regulatory networks is essential for understanding cellular functions.
• Existing methods are limited to single RBP analysis and lack single-molecule resolution.

Purpose of the Study:

• To introduce TRIBE-STAMP, a method for concurrent identification of RNA targets for two RBPs.
• To provide a detailed protocol for TRIBE-STAMP, including computational analysis.
• To enable single-molecule resolution for RBP target identification.

Main Methods:

• TRIBE-STAMP involves fusing RBPs to RNA editing enzymes (ADAR or APOBEC1).
• RNA sequencing (RNA-seq) is performed on cells expressing these fusion proteins.
• Computational analysis identifies A-to-I and C-to-U editing events to pinpoint RBP-bound RNAs.

Main Results:

• TRIBE-STAMP enables simultaneous identification of RNA targets for two distinct RBPs.
• The method achieves single-molecule resolution, overcoming limitations of previous techniques.
• The protocol details fusion protein expression and data analysis for TRIBE-STAMP.

Conclusions:

• TRIBE-STAMP is a versatile and simple approach for identifying RBP targets.
• This method offers unprecedented insights into the interplay between pairs of RBPs.
• TRIBE-STAMP advances the study of RNA-binding protein functions and regulatory networks.