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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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Published on: May 10, 2018

A conserved PUF-Ago-eEF1A complex attenuates translation elongation.

Kyle Friend¹, Zachary T Campbell, Amy Cooke

¹Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Nature Structural & Molecular Biology

|January 11, 2012

Summary

A newly discovered complex of Pumilio (PUF) and Argonaute (Ago) proteins with translation factor eEF1A represses gene expression by inhibiting translation elongation. This conserved mechanism was observed in both nematodes and mammals.

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Area of Science:

Molecular Biology
Gene Regulation
Protein Complexes

Background:

Pumilio (PUF) and Argonaute (Ago) proteins are key regulators of mRNA post-transcriptional control.
These proteins utilize distinct yet similar mechanisms to influence gene expression.

Purpose of the Study:

To investigate the potential for PUF and Ago proteins to function together in a complex.
To identify novel roles for PUF-Ago complexes in post-transcriptional gene regulation, specifically in translation elongation.

Main Methods:

Co-immunoprecipitation assays were used to identify PUF-Ago-eEF1A complexes in both nematode (Caenorhabditis elegans) and mammalian systems.
Recombinant protein assays were employed to study the functional interactions within these complexes.
In vivo assays in C. elegans assessed the repression of PUF target mRNAs.
In vitro assays examined the effect of mammalian PUF-Ago-eEF1A complexes on reporter mRNA translation and eEF1A GTPase activity.

Main Results:

Conserved PUF-Ago-eEF1A complexes were identified in both nematodes and mammals.
Nematode CSR-1 (Ago) facilitated the repression of FBF (PUF) target mRNAs in vivo.
The FBF-1-CSR-1 heterodimer inhibited the GTPase activity of EFT-3 (eEF1A) in vitro.
Mammalian PUM2-Ago-eEF1A complexes repressed translation of reporter mRNAs in vitro, independent of polyadenylation.
Repression was observed post-initiation, leading to ribosome accumulation within the open reading frame.

Conclusions:

A conserved PUF-Ago-eEF1A complex functions to attenuate translation elongation.
This complex represents a novel mechanism for post-transcriptional gene regulation.
The findings reveal a coordinated role for RNA-binding proteins and translation factors in controlling gene expression at the elongation step.