Tho2 sequences from S. cerevisiae, D. melanogaster, D. rerio, M. musculus, and H. sapiens were aligned with ClustalW. Shading indicates the degree of conservation across homologues. Tho2 secondary structural features from our cryo-EM structure are shown above the sequence alignment with α-helices represented as blue bars and β-sheets represented as blue arrows. The model of Tho2 contains 10 poly-Ala helices at its C-terminus represented by gray line. Three poly-Ala helices at the N-terminus of Tho2 are represented by gray bars. Sequences that are not present in the model (loops and highly flexible regions) are represented by dotted lines. The regions of Tho2 that form interaction interfaces with each of the other THO complex subunits were identified using PISA, and these interaction regions are shown below the sequence alignment. The coloration for the interaction regions is consistent with the color scheme throughout the manuscript with Hpr1 in green, Tex1 in cyan, Mft1 in light blue, Thp2 in yellow, Sub2-N in pink, and Sub2-C in purple.