Cross-linking mass spectrometric analysis of the endogenous TREX complex fromSaccharomyces cerevisiae

Abstract

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3′ end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. InSaccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex ofS. cerevisiaepurified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO–Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.