ALYREF directly binds to CBC.

(A) Schematics of the NCBP1 and NCBP2 subunits of the human CBC and ALYREF. ALYREF contains a central RRM domain. The RRM domain is connected to two conserved motifs (UBMs) at both termini through variable regions (N-vr and C-vr). (B) ALYREF directly interacts with the CBC. In vitro GST pull down assays were performed with purified recombinant human CBC and GST-tagged ALYREF (residues 1-183) or mALYREF2 (residues 1-155). Results are representative of three independent experiments.

Cryo-EM structures of CBC and CBC-mALYREF2

(A) Overall architecture of the CBC complex. NCBP1 and NCBP2 are colored in yellow and teal, respectively. The cap analog is shown as orange sticks. (B) Comparison of the CBC cryo-EM structure to the unliganded CBC crystal structure (PDB ID 1N54). The cryo-EM structure is colored as in (A). Unliganded CBC crystal structure is colored in light blue. Arrow indicates the conformational change in the N-terminal extension of NCBP2 upon cap analog binding. (C) Overall architecture of the CBC-mALYREF2 complex. NCBP1, NCBP2, and mALYREF2 are colored in yellow, teal, and red, respectively. The RRM domain of mALYREF2 binds to the CBC. (D) Comparison of the CBC-mALYREF2 and the CBC cryo-EM structures. CBC structure is colored in gray. Arrow indicates conformational changes in a loop (residues 38-45) of NCBP1 upon mALYREF2 binding.

Cryo-EM data collection, refinement and validation statistics

ALYREF binds to both NCBP1 and NCBP2.

(A) Sequence alignment of the RRM domain of mALYREF2 and ALYREF62. The residues that interface with the CBC are indicated by triangles below the sequence. The triangles colored in purple correspond to residues subjected to mutagenesis in Figure 4. (B) Details of the interaction between NCBP1 and mALYREF2. As a reference, an overall model is shown on the top to indicate the zoomed-in area. (C) Details of the interaction between NCBP2 and mALYREF2. (D) mALYREF2 features a positively charged surface near the cap analog bound to NCBP2. Surface of mALYREF2 is colored according to the electrostatic potential, ranging from red (−5 kBT/e) to blue (+5 kBT/e). Dotted line indicates putative RNA binding path.

Dissection of the ALYREF and CBC interfaces.

(A) Mutations of key interface residues on ALYREF reduced CBC binding. In vitro GST pull down assays were performed with purified recombinant human CBC and GST-tagged ALYREF-RRM wild type or mutants (mut-1, Y166R/V169R/P170R; mut-2, E124R/E128R). (B) NCBP1 is sufficient to interact with ALYREF. In vitro GST pull down assays were performed with purified recombinant GST-tagged ALYREF and individual CBC subunits. Results are representative of three independent experiments.

Functional implications for ALYREF and CBC in 5’ cap dependent mRNP export.

(A) ALYREF links the TREX complex to the CBC. The RRM domain of ALYREF recognizes the CBC at the 5’ end of mRNA. The UBM of ALYREF binds to the DDX39B subunit of TREX; their complex is represented using their yeast orthologs Yra1 and Sub2, respectively (PDB ID 5SUP). DDX39B in turn associates with the THO subcomplex of TREX. (B) HVS ORF57 binds to the RRM domain of ALYREF and interferes with the CBC-ALYREF interaction. (left) NMR structure of the ALYREF-ORF57 complex (PDB ID 2YKA). (right) The CBC-ALYREF structure is overlayed with the ALYREF-ORF57 structure. (C) Model of viral mRNA export mediated by herpes viral ORF57 homologs. On viral transcripts, ALYREF associates with the CBC and ORF57 via the WxHD motif and the RRM domain, respectively. Both ALYREF and ORF57 feature RNA binding regions to form contacts with the RNA. ALYREF recruits the other TREX complex components to facilitate nuclear export of viral mRNAs.

ALYREF and CBC in splicing and export

(A) Overlay of the CBC-ALYREF structure with the CBC-SRSF1 structure (PDB ID 7ABG). CBC-ALYREF is colored as in Figure 2B. CBC-SRSF1 is colored with the CBC in white and SRSF1 in orange. (B) ALYREF interaction with the CBC and the EJC is mutually exclusive. (left) ALYREF binds to the MAGOH subunit of the EJC (PDB ID 7ZNJ). (right) The CBC-ALYREF structure is overlayed with the ALYREF-MAGOH structure. (C) Model of the mRNP export receptor NXF1-NXT1 recruitment by CBC-ALYREF and other factors during mRNA maturation.

Human ALYREF and mouse ALYREF2 are conserved.

Sequence alignment of ALYREF and mALYREF262. The DDX39B binding motifs (N-UBM and C-UBM), the WxHD motif, and the RRM domain are indicated.

Workflow for cryo-EM data processing.

Local resolution estimation and FSC curves.

(A) Local resolution of the CBC density map. (B) FSC curves between the two half maps of CBC. (C) Local resolution of the CBC-mALYREF2 density map. (D) FSC curves between the two half maps of CBC-mALYREF2. (E) Electron density map of CBC-mALYREF2 at the interface between NCBP1 and mALYREF2. (F) Electron density map of CBC-mALYREF2 at the interface between NCBP1 and NCBP2.

HSV-1 ICP27 binds to the RRM domain of ALYREF and interferes with the CBC-ALYREF interaction.

(A) NMR structure of the ALYREF-ICP27 complex (PDB ID 2KT5). (B) The CBC-ALYREF structure is overlayed with the ALYREF-ICP27 structure.