Protocol for in vitro transcribing mRNAs with defined poly(A)-tail lengths and visualizing sequential PABP binding

TL;DR

This paper provides a detailed protocol for synthesizing mRNAs with specific poly(A)-tail lengths and observing how proteins bind to them in a cell-free system.

Contribution

A novel PCR-based method for creating mRNAs with defined poly(A) tails and a cell-free system to visualize PABPC binding.

Findings

A PCR-based approach allows precise synthesis of mRNAs with specific poly(A)-tail lengths.

Quality control steps ensure the reliability of poly(A)-tailed mRNA products.

An in vitro method visualizes sequential binding of PABPCs to poly(A) tails.

Abstract

Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure.

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

•In vitro synthesis of mRNAs with defined poly(A)-tail length•Quality control procedures for both poly(A)-tailed PCR and mRNA products•In vitro protocol for the visualization of PABPCs’ binding to the poly(A) tail

In vitro synthesis of mRNAs with defined poly(A)-tail length

Quality control procedures for both poly(A)-tailed PCR and mRNA products

In vitro protocol for the visualization of PABPCs’ binding to the poly(A) tail

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

Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of PABPCs to these poly(A) tails. We detail quality control steps throughout the procedure.

Figures