Extended Data Fig. 3: Comparisons of ALYREF–EJC interaction details with a viral ALYREF–ORF57 complex, the cytoplasmic CASC3–EJC–RNA complex, and the EJC-bound P-complex spliceosome.
From: mRNA recognition and packaging by the human transcription–export complex
a. Organization of ALYREF. Top: Structural model of full-length ALYREF predicted with AlphaFold84,85. Annotated domains (N-UBM, WxHD motif, RRM and C-UBM) are colored in darker shades of purple. Spheres represent backbone atoms of glycine and arginine residues in the RBD domains. Middle: ALYREF domain diagram. Black bars indicate residues that are included as an atomic model in this study. Bottom: AlphaFold per residue confidence score (pLDDT) plot. High values are indicative of high confidence predictions, whereas low values represent residues that are likely disordered in solution. b. Comparison of the ALYREF RRM domain interaction with the EJC subunit MAGOH (interface c, left) and the Herpes simplex virus ORF57 (right)33. ALYREF binds viral ORF57 differently compared to the overlapping ALYREF–EJC interface c. This supports a general model that ALYREF can use multiple interfaces to engage either viral proteins, such as ORF57, or mRNP maturation marks, such as the CBC or EJCs, and may enable ALYREF to broadly select its RNA targets. c. Details of the WxHD motifs binding to the EJC. Left: Modelling of apo EIF4A3 bound to the WxHD motif indicates a clash with EIF4A3 residue Y202, suggesting that ALYREF can only bind to RNA-bound EJC (see Supplementary Video 2). Middle: the same view, showing the ALYREF WxHD motif bound to RNA-bound EJC (this study). Right: the same view, showing the CASC3 WxHD motif bound to RNA-bound EJC23, revealing conserved binding modes of ALYREF and CASC3. d. The ALYREF WxHD and RRM domains binds the same interfaces between EIF4A3 and MAGOH as the CASC3SELOR domain. Top: Overview image of the ALYREF–EJC–RNA structure (left) and comparison of the binding modes of ALYREF and CASC3 (middle and right, respectively). Bottom: Sequence alignment of ALYREF (top) and CASC3 (bottom), showing the conserved WxHD motif and an additional short conserved motif (QEL[F/I]Ax[F/Y]G), which is however not contacting the EJC in the ALYREF–EJC–RNA structure. Conserved (dark blue) and partially conserved (light blue) residues are indicated with boxes. Residues in ALYREF and CASC3 contacting the EJC are indicated. e. Superposition of the ALYREF–EJC–RNA complex (this study) onto the human P-complex spliceosome cryo-EM structure (PDB ID 6QDV)32, via their EJC EIF4A3 subunits. This model reveals that higher order ALYREF–EJC complexes such as the ALYREF–EJC dimer are not possible when the EJC is still bound by the spliceosomes, as the P-complex subunit SNU114 clashes with the RRM in an ALYREF–EJC dimer. In addition, SNU114 likely disfavors binding of a single molecule of ALYREF to the EJC, as there is a steric clash with the N-terminal ordered ALYREF residue (Asp 85) in the ALYREF–EJC structure.
