Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

John M Pagano,Brian M Farley,Lisa M Mccoig,Sean P Ryder

doi:10.1074/jbc.m700079200

John M Pagano, Brian M Farley + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m700079200

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Abstract

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9-13-nucleotide window. This sequence is remarkably abundant in the 3'-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation.

Highlights

In the nematode worm Caenorhabiditis elegans, polarization of the body axes occurs after fertilization and requires several highly conserved maternal factors termed PAR proteins [3,4,5,6,7,8,9,10]
MEX-5 and MEX-6 Bind to ARE Repeat Elements—To clarify the role of MEX-5 and MEX-6 in development, we set out to test whether these proteins, like TTP, bind with high affinity and specificity to RNA
A recombinant fragment of each protein comprising the tandem zinc finger (TZF) domain was purified from bacteria and tested for the ability to interact with an established TTP binding sequence, the AU-rich element (ARE) of TNF-␣ mRNA [26]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression Constructs—Fragments of mex-5 and mex-6 containing the TZF domain (amino acids 236 –350 and 250 – 400, respectively) were amplified from ORFeome clones (Open Biosystems) and subcloned into the vector pMal-c (New England Biolabs). Fractions containing the fusion protein were pooled and dialyzed into Q buffer (50 mM Tris, pH 8.8, 20 mM NaCl, 2 mM dithiothreitol, and 100 ␮M Zn(OAc)2) and further purified over a Hi-trap Q HP column (GE Healthcare). Final purification was achieved by combining fractions containing the protein and dialyzing them into S buffer (50 mM MOPS, pH 6.0, 20 mM NaCl, 2 mM dithiothreitol, 100 ␮M Zn(OAc)2) before running them over a Hi-trap S HP column (GE Healthcare). Typical reactions consisted of 2– 4 nM labeled RNA equilibrated with varying concentrations of protein in equilibration buffer for 3 h. The elevated concentration of RNA enables determination of the apparent stoichiometry by measuring the equivalent saturation point This value was estimated by plotting polarization as a function of molar equivalents of protein to RNA and performing linear fits to pre- and post-saturation data. DIC and GFP images were collected with live specimens using a Zeiss Axioskop microscope with ϫ40 or 100 objectives

RESULTS

A Single Residue in Each Finger

DISCUSSION