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The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators.

Elisha Mugo¹, Esteban D Erben²

¹Department of Biochemistry, Genetics and Microbiology, Institute for Sustainable Malaria Control, University of Pretoria, Hatfield, South Africa.

Methods in Molecular Biology (Clifton, N.J.)

|March 30, 2020

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Researchers developed a tethering assay to identify novel RNA-binding proteins (RBPs) that regulate gene expression in Trypanosoma brucei. This method allows functional analysis of RBPs independently of their RNA-binding ability, revealing new mRNA fate regulators.

Area of Science:

Molecular Biology
Genetics
Parasitology

Background:

Posttranscriptional control is crucial for gene expression regulation in trypanosomatids.
RNA-binding proteins (RBPs) orchestrate these controls by interacting with RNA regulatory elements.
Understanding RBP function is key to deciphering gene regulation in these organisms.

Purpose of the Study:

To introduce and validate a robust tethering assay for functional analysis of RBPs in Trypanosoma brucei.
To identify novel mRNA-fate regulators through a high-throughput screening approach.
To provide a detailed protocol for the tethering assay applicable in various research settings.

Main Methods:

The tethering assay involves fusing a protein of interest to an mRNA reporter via an artificial RNA-protein interaction.

Keywords:

Lambda N-peptide RNA stability RNA-binding proteins Tethering Translation mRNA fate

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A Trypanosoma brucei cell line expressing a chloramphenicol acetyltransferase (CAT) reporter mRNA was utilized.

Functional impact was assessed by measuring CAT activity after induction of the fusion protein.

Main Results:

The tethering assay successfully enabled the functional characterization of RBPs independent of their intrinsic RNA-binding capabilities.
Dozens of novel mRNA-fate regulators were identified in cultured Trypanosoma brucei.
The assay demonstrated simplicity and efficiency, with analysis typically completed within one working day per protein.

Conclusions:

The tethering assay is a powerful and accessible tool for discovering and functionally characterizing RNA-binding proteins in trypanosomatids.
This method significantly advances the study of posttranscriptional gene regulation in Trypanosoma brucei.
The described protocol facilitates the widespread adoption of this technique for identifying novel gene expression regulators.