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

Carmen Grandi; Martin Emmaneel; Frank H T Nelissen; Maike M K Hansen

doi:10.1016/j.xpro.2024.103284

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

STAR Protoc. 2024 Sep 20;5(3):103284. doi: 10.1016/j.xpro.2024.103284. Epub 2024 Aug 31.

Authors

Carmen Grandi¹, Martin Emmaneel¹, Frank H T Nelissen¹, Maike M K Hansen²

Affiliations

¹ Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands.
² Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands; Oncode Institute, Nijmegen, the Netherlands. Electronic address: maike.hansen@ru.nl.

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.

Keywords: Bioinformatics; Cell-based Assays; Gene Expression; Molecular Biology.

MeSH terms

Biochemistry* / methods
Humans
Poly A* / metabolism
Poly(A)-Binding Proteins* / genetics
Poly(A)-Binding Proteins* / metabolism
Polymerase Chain Reaction / methods
Protein Binding
RNA, Messenger* / genetics
RNA, Messenger* / metabolism
Transcription, Genetic / genetics

Substances

Poly A
Poly(A)-Binding Proteins
RNA, Messenger