Structural characterization of the principal mRNA-export factor Mex67-Mtr2 from Chaetomium thermophilum.

Shintaro Aibara,Lyudmila Dimitrova,Murray Stewart,Meindert H Lamers,Ed Hurt,Eugene Valkov

doi:10.1107/s2053230x15008766

Abstract

Members of the Mex67-Mtr2/NXF-NXT1 family are the principal mediators of the nuclear export of mRNA. Mex67/NXF1 has a modular structure based on four domains (RRM, LRR, NTF2-like and UBA) that are thought to be present across species, although the level of sequence conservation between organisms, especially in lower eukaryotes, is low. Here, the crystal structures of these domains from the thermophilic fungus Chaetomium thermophilum are presented together with small-angle X-ray scattering (SAXS) and in vitro RNA-binding data that indicate that, not withstanding the limited sequence conservation between different NXF family members, the molecules retain similar structural and RNA-binding properties. Moreover, the resolution of crystal structures obtained with the C. thermophilum domains was often higher than that obtained previously and, when combined with solution and biochemical studies, provided insight into the structural organization, self-association and RNA-binding properties of Mex67-Mtr2 that facilitate mRNA nuclear export.

Highlights

The Mex67–Mtr2 complex (NXF1–NXT1 in metazoans and small bristles in Drosophila melanogastor) is the principal mRNA-export factor in Sacccharomyces cerevisiae (Segref et al, 1997; Herold et al, 2000; Wilkie et al, 2001)
Mex67/NXF1 has a modular structure based on four domains (RRM, leucine-rich repeat (LRR), NTF2-like and UBA) that are thought to be present across species, the level of sequence conservation between organisms, especially in lower eukaryotes, is low
Fragments corresponding to the RNA-recognition motif (RRM) and LRR domains were cloned into the first multiple cloning site (MCS) of pETDuet-1 to generate His6-tagged constructs

Summary

Introduction

The Mex67–Mtr complex (NXF1–NXT1 in metazoans and small bristles in Drosophila melanogastor) is the principal mRNA-export factor in Sacccharomyces cerevisiae (Segref et al, 1997; Herold et al, 2000; Wilkie et al, 2001). The viral RNA CTE (constitutive transport element) sequence motif (Pasquinelli et al, 1997; Gruter et al, 1998; Braun et al, 1999; Wiegand et al, 2002; Teplova et al, 2011) Both the NTF2L domain complexed with Mtr2/NXT1 and the UBA domain interact with a range of FG nucleoporins (Fribourg et al, 2001; Grant et al, 2003). We present the crystal structures of the individual domains of C. thermophilum Mex67–Mtr (ctMex67–Mtr2) to provide a complete repertoire of high-resolution structures from a single species to facilitate structural studies of the complexes formed during the formation of export-competent mRNPs. In vitro SAXS and RNA-binding studies have demonstrated that ctMex67–Mtr has similar biochemical properties as H. sapiens NXF1–NXT1 (hsNXF1–NXT1) and, in particular, have shown that the ctMex NTF2L domain contributes to mRNA binding

Cloning and protein purification

X-ray crystallography

Small-angle X-ray scattering

In vitro RNA-binding assays

Results and discussion

RNA binding of ctMex67–Mtr2