Protocol to distinguish pre-mRNA from mRNA in RNA-protein interaction studies

TL;DR

This paper introduces a protocol to determine whether RNA-binding proteins prefer binding to pre-mRNA or mRNA, using tristetraprolin as a model.

Contribution

A novel protocol to distinguish RBP binding preferences for pre-mRNA versus mRNA is presented.

Findings

The protocol enables quantification of intronic and exonic RNA fragments bound to RBPs.

The method can be applied to any RNA-binding protein to assess binding preferences.

Steps for pull-down, genome processing, and intron-exon ratio calculation are detailed.

Abstract

Transcriptome-wide studies on interactions between RNA-binding proteins (RBPs) and protein-coding RNAs in general preclude interpretations regarding RBP preference for binding to the more abundant mRNA over the less abundant pre-mRNA. Here, we present a protocol to determine the binding preference of the RBP tristetraprolin (TTP, Zfp36) for pre-mRNA versus mRNA. We describe steps for the identification and quantitation of intronic and exonic fragments in RNA bound to TTP. This protocol can potentially be applied to any RBP.

For complete details on the use and execution of this protocol, please refer to Bestehorn et al.1

•Steps for efficient pull-down of full-length RNAs bound to RBPs•Reference genome processing and unambiguous intronic and exonic fragment counting•Intron-exon ratio calculation and determination of RBP binding preference

Steps for efficient pull-down of full-length RNAs bound to RBPs

Reference genome processing and unambiguous intronic and exonic fragment counting

Intron-exon ratio calculation and determination of RBP binding preference

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Transcriptome-wide studies on interactions between RNA-binding protein (RBPs) and protein-coding RNAs in general preclude interpretations regarding RBP preference for binding to the more abundant mRNA over the less abundant pre-mRNA. Here, we present a protocol to determine the binding preference of the RBP tristetraprolin (TTP, Zfp36) for pre-mRNA versus mRNA. We describe steps for the identification and quantitation of intronic and exonic fragments in RNA bound to TTP. This protocol can potentially be applied to any RBP.

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