(a) Domain organization of human ALYREF (yeast Yra1). UBM, UAP56 Binding Motif; RBD, RNA Binding Domain; RRM, RNA-Recognition Motif. The RBD contains multiple RG- motifs. (b) Amino acid sequence alignment comparing N-terminal (left) and C-terminal (right) UBMs of ALYREF from Homo sapiens (Hs), Danio rerio (Dr), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Arabidopsis thaliana (At), Saccharomyces cerevisiae (Sc), and Schizosaccharomyces pombe (Sp). The alignment was created using Clustal Omega (Sievers et al., 2011) and visualized with ESPript 3 (Robert and Gouet, 2014). A secondary structure assignment, based on PSIPRED (Buchan et al., 2013) for the N-UBM and the crystal structure of Yra1 C-UBM bound to Sub2 (PDB ID 5SUP) for the C-UBM, is shown above the alignment. Purple stars indicate residues predicted to not bind the UAP56 RecA1 lobe. Invariant and conserved residues are indicated with a red background or red font, respectively. (c) Amino acid sequence alignment as in panel b but comparing N-UBMs of human ALYREF, POLDIP3, UIF and LUZP4 on the top, and the C-UBMs of human ALYREF and CHTOP below. (d) Principal component analysis (PCA) of THO–UAP56 cryo-EM data reveals flexibility. Densities for the start (green) and endpoint (gray) of the first two PCA components are shown from front and left side views. Component two is additionally shown from a top view, as in panel e. Dimers 1 and 2, monomers A and B, and the pseudo two-fold symmetry axis are indicated. (e) Modeling of THO–UAP56 monomers 1A and 2B into densities from PCA component two shows that proximal UAP56 helicases approach each other (ribbon model, colored as in Figure 2). Conformation two is the conformation of the THO–UAP56 structure, while conformations 1 and 3 reflect the starting and endpoints for the PCA component two movement shown in panel d. The UAP56 RecA1 lobes are not resolved in our structure and are indicated as dashed circles. The inset shows a surface representation of the THO–UAP tetramer, colored as in Figure 3a, with the subunits shown below depicted as ribbons. THOC5, −6 and −7 were omitted for clarity. (f) Details of the TREX–mRNA complex model shown in Figure 4b (ribbon model, colored as in Figure 4b). Side chains of ALYREF C-UBM residues in stick representation, with conserved acidic residues labelled. Conserved, basic residues on the neighboring UAP562B RecA2 lobe are shown as gray sticks and are labelled. (g) The DEXD-box RNA helicase RecA2 lobe is frequently bound by other proteins. Surface view of RNA-bound UAP56 (homology model based on the Sub2–Yra1–RNA crystal structure [Ren et al., 2017] [PDB ID 5SUP]), with RNA shown as ribbons. RecA1 lobe (purple), RecA2 lobe (pink), RNA (black) and UAP56 RecA2 lobe residues highlighted in panel f (gray). Superimposed on UAP56, and shown as Cα trace, are Edc3 (residues 88–116, Dhh1-bound conformation, PDB ID 4BRU), 4ET (residues 216–238, DDX6-bound conformation, PDB ID 5ANR), DDX6 C-terminus (residues 461–469, PDB ID 4CT4) and CWC22 (residues 131–136, eIF4AIII-bound conformation, PDB ID 4CB9). Other DEXD-box helicases, except for UAP56, were omitted for clarity.