Figures
Figure 1 with 1 supplement
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 …
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Figure 1—source data 1
Original file for the gels in Figure 1B.
- https://cdn.elifesciences.org/articles/91432/elife-91432-fig1-data1-v1.zip
Figure 1—figure supplement 1
Human ALYREF and mouse ALYREF2 are conserved.
Sequence alignment of ALYREF and mALYREF2 (Robert and Gouet, 2014). The DDX39B binding motifs (N-UBM and C-UBM), the WxHD motif, and the RRM domain are indicated.
Figure 2 with 5 supplements
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 …
Figure 2—figure supplement 1
Workflow for cryo-EM data processing.
Data were processed in CryoSPARC. A set of 241,915 particles yielded a reconstruction of a CBC-mALYREF2 map at 3.22 Å resolution. A set of 78,039 particles yielded a reconstruction of a CBC map at …
Figure 2—figure supplement 2
Cryo-EM reconstruction of the CBC.
(A) Selective 2D class averages from the final particle set of the CBC. (B) The gold-standard FSC curves of the final reconstruction of the CBC. (C) Angular distribution of the final reconstruction …
Figure 2—figure supplement 3
Structural model of the CBC.
(A) Map versus model FSC curves with or without mask calculated using Phenix. A resolution of 3.6 Å is estimated at FSC = 0.5. (B, C) Electron density map and structural model of CBC at the …
Figure 2—figure supplement 4
Cryo-EM reconstruction of the CBC-mALYREF2 complex.
(A) Selective 2D class averages from the final particle set of CBC-mALYREF2. (B) The gold-standard Fourier shell correlation (FSC) curves of the final reconstruction of CBC-mALYREF2. (C) Angular …
Figure 2—figure supplement 5
Structural model of the CBC-mALYREF2 complex.
(A) Map versus model Fourier shell correlation (FSC) curves with or without mask calculated using Phenix. A resolution of 3.5 Å is estimated at FSC = 0.5. (B, C) Electron density map and structural …
Figure 3 with 1 supplement
ALYREF binds to both NCBP1 and NCBP2.
(A) Sequence alignment of the RRM domain of mALYREF2 and ALYREF (Robert and Gouet, 2014). The residues that interface with the CBC are indicated by triangles below the sequence. The triangles …
Figure 3—figure supplement 1
Electron density maps at the CBC-mALYREF2 interfaces.
Figure 4
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 …
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Figure 4—source data 1
Original file for the gels in Figure 4A.
- https://cdn.elifesciences.org/articles/91432/elife-91432-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for the gels in Figure 4B.
- https://cdn.elifesciences.org/articles/91432/elife-91432-fig4-data2-v1.zip
Figure 5 with 1 supplement
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 …
Figure 5—figure supplement 1
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.
Figure 6
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 …
Tables
Table 1
Cryo-EM data collection, refinement, and validation statistics.
| CBC-mALYREF2(EMDB EMD-40739)(PDB 8SRR) | CBC(EMDB EMD-40780)(PDB 8SUY) | |
|---|---|---|
| Data collection and processing | ||
| Microscope/camera | Glacios/Falcon 4i | |
| Voltage (kV) | 200 | |
| Electron exposure (e–/Å2) | 52 | |
| Defocus range (μm) | –1.0 to –2.0 | |
| Pixel size (Å) | 0.732 | |
| Box size (pixels) | 288 | |
| Initial particle images (no.) | 1,625,826 | |
| Final particle images (no.) | 241,915 | 78,039 |
| Map resolution (masked, Å) | 3.22 | 3.38 |
| Fourier shell correlation (FSC) threshold | 0.143 | 0.143 |
| Refinement | ||
| Model resolution (masked, Å) | 3.5 | 3.6 |
| FSC threshold | 0.5 | 0.5 |
| Model composition | ||
| Protein residues | 975 | 895 |
| Ligands | 1 | 1 |
| B factors (Å2) | ||
| Protein | 162.9 | 180.1 |
| Ligand | 157.5 | 163.2 |
| r.m.s. deviations | ||
| Bond lengths (Å) | 0.003 | 0.003 |
| Bond angles (°) | 0.417 | 0.413 |
| Validation | ||
| MolProbity score | 1.34 | 1.48 |
| Clashscore | 6.24 | 7.37 |
| Poor rotamers (%) | 0.7 | 0.9 |
| Ramachandran plot | ||
| Favored (%) | 98.0 | 97.6 |
| Allowed (%) | 2.0 | 2.4 |
| Disallowed (%) | 0.0 | 0.0 |
