Structural and functional insights into TREX complex interactions

Abstract

Gene expression in eukaryotes involves mRNA synthesis, processing, and export from the nucleus to the cytoplasm. The highly conserved TREX (transcription/export) complex plays a crucial role in coupling these processes, ensuring the efficient transition of mRNA from transcription to translation (Sträßer et al. 2002). Since only limited research was available on the structural organization of the TREX complex, the first objective was to investigate the endogenous TREX complex structure from Saccharomyces cerevisiae. The second objective was to analyze the structure of the Sub2-Yra1-Tho1 subcomplex from Chaetomium thermophilum. Tho1 has been suggested to function in transcription, splicing, export, and/or translation (Aravind & Koonin 2000; Hashii et al. 2004; Leaw et al. 2004). In addition to the structural objectives, this study also aimed to gain information of Tho1's functions in C. thermophilum. This research showed the predominantly dimeric arrangement of the complete endogenous S. cerevisiae TREX complex, determined using XL-MS and EM. While the flexible C-terminus of yeast Tho2 is not essential for the formation of TREX, it is critical for cellular functions, particularly under stress conditions. Co-expressing the C. thermophilum proteins ctSub2, ctTho1, and ctYra1 in Escherichia coli enabled the purification of a stable ctSub2-ctTho1-ctYra1 (S-Y-T) complex. Despite nuclease treatment during the purification, this complex retained its association with nucleic acids. Cryo-EM images of the nuclease-treated S-Y-T complex revealed larger complexes, approximately 20 nm in size, tightly bound to RNA. Especially the interaction between ctSub2 and ctTho1 was highly stable, which allowed the successful in vitro reconstitution of this protein-protein complex. Further in vitro reconstitution experiments with various ctTho1 deletion mutants revealed that the C-terminal domain (CTD) of ctTho1, which includes two DIM motifs, is crucial for interacting with ctSub2. Although the C-terminal end (CTE) of ctTho1, which contains an additional DIM motif, is not essential, it enhances the ctSub2 interaction. Moreover, the flexible CTE carries out important functions of ctTho1, such as binding and annealing different RNA and DNA oligos. In conclusion, this study shows the predominantly dimeric organization of the endogenous yeast TREX complex and highlights the importance of structurally unresolved, flexible protein regions – such as the yeast Tho2 C-terminus or the ctTho1 CTE – in maintaining cellular functionality. Given that ctTho1 has three DIM motifs, the S-Y-T complex in C. thermophilum likely contains multiple ctSub2 molecules. By interacting with the alpha-helical N- and C-box regions of ctYra1, the ctSub2 proteins are possibly compacted by several ctYra1 proteins, resulting in a multiprotein S-Y-T complex.