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. 2024 Sep 16;12:RP91432. doi: 10.7554/eLife.91432

Cryo-EM structure of the CBC-ALYREF complex

Bradley P Clarke 1, Alexia E Angelos 1, Menghan Mei 1, Pate S Hill 1, Yihu Xie 1,✉, Yi Ren 1,2,✉
Editors: Yang Yang3, Amy H Andreotti4
PMCID: PMC11405014  PMID: 39282949

Abstract

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

Research organism: Human

Introduction

The nuclear cap binding complex (CBC) binds to the m7G cap of RNAs transcribed by RNA pol II. It is comprised of NCBP1 (also known as CBP80) and NCBP2 (also known as CBP20). This heterodimeric CBC can form a variety of interactions with different proteins to promote mRNA processing and influence the fate of the transcript. As such, the CBC regulates gene expression at multiple levels, ranging from transcription and splicing to nuclear export and translation (Gonatopoulos-Pournatzis and Cowling, 2014; Rambout and Maquat, 2020).

The CBC is one of a myriad of protein factors that associate with newly synthesized transcripts. These factors package mRNAs into compacted ribonucleoprotein particles (mRNPs). While the overall structural arrangement of mRNPs is not known, factors such as serine/arginine-rich (SR) proteins are suggested to contribute to mRNP compaction. Prior to export, mRNPs acquire the export receptor NXF1-NXT1 to gain access to the nuclear pore complex (NPC) (Xie and Ren, 2019). NXF1-NXT1 can interact with the FG repeats of the nucleoporin proteins in the NPC to mediate mRNA export (Fribourg et al., 2001; Grant et al., 2003). Intriguingly, early electron microscopic studies on the Balbiani ring mRNPs in Chironomus tentans showed that mRNPs translocate through the NPC in a 5′ to 3′ direction (Mehlin et al., 1992). Recent single-molecule work on human mRNPs also suggests mRNA is exported 5′ end first (Ashkenazy-Titelman et al., 2022).

The CBC promotes nuclear mRNA export through its interaction with a key mRNA export factor ALYREF. ALYREF in turn binds to DDX39B (also known as UAP56) and by extension, the entire TRanscription-EXport (TREX) complex at the 5′ end of the RNA (Cheng et al., 2006). The TREX complex is conserved from yeast to humans (Strässer et al., 2002; Ren et al., 2017; Xie et al., 2021a; Pacheco-Fiallos et al., 2023; Schuller et al., 2020). The human TREX complex is composed of THOC1, 2, 3, 5, 6, 7, and the DEAD-box helicase DDX39B (Masuda et al., 2005). Interestingly, ALYREF was also identified as the THOC4 subunit of the TREX complex, indicating that the function of ALYREF is tightly integrated with the TREX complex. ALYREF and its yeast ortholog Yra1 contain UBMs (UAP56-binding motifs) that mediate the interaction with DDX39B and Sub2, respectively (Ren et al., 2017; Luo et al., 2001; Chang et al., 2013; Strässer and Hurt, 2001). The yeast CBC was also shown to facilitate the recruitment of Yra1 onto nascent RNA (Sen et al., 2019). ALYREF and TREX play central roles in mRNA export through direct interactions with various factors including the export receptor NXF1-NXT1 (Viphakone et al., 2012; Strässer and Hurt, 2000; Stutz et al., 2000). Their association with the 5′ cap of mRNAs in particular is a key step in the export process. However, thus far the molecular mechanism underlying how ALYREF bridges the CBC and TREX remains unclear.

ALYREF and the associated TREX complex are not only required for cellular mRNA export but also can be hijacked for nuclear export of some viral mRNAs, such as those of herpes viruses. Two well-studied examples are Herpes simplex virus (HSV-1) ICP27 and Herpesvirus saimiri (HVS) ORF57 (Boyne et al., 2008; Sandri-Goldin, 2008). Both HSV-1 ICP27 and HVS ORF57 directly target the host ALYREF protein (Tunnicliffe et al., 2011; Tunnicliffe et al., 2014). Structural studies show that they recognize overlapping surfaces on the RRM domain of ALYREF. How these viral factors affect host CBC-ALYREF interaction and function is not known.

The highly integrated nuclear mRNA processing and mRNP packaging also require the actions of multi-functional splicing factors, such as the SR protein SRSF1 and the exon junction complex (EJC). SRSF1 couples transcription, splicing, and export through direct interactions with the CBC, spliceosome, NXF1-NXT1, and RNA (Huang et al., 2004; Cléry et al., 2013; Townsend et al., 2020). Of note, ALYREF also has binding activities to these factors. Therefore, the functions of ALYREF and SRSF1 in mRNA processing and export are likely interconnected. In higher eukaryotes, the EJC is deposited 20–24 nucleotides upstream of the exon junctions during splicing (Le Hir et al., 2000; Andersen et al., 2006; Bono et al., 2006). The EJC was initially shown to associate with ALYREF through a WxHD motif in the N-terminal unstructured region of ALYREF (Gromadzka et al., 2016). More recently, multiple binding interfaces were shown between ALYREF and the EJC (Pacheco-Fiallos et al., 2023). How the CBC-ALYREF connection affects the function of EJC-ALYREF remains elusive.

To better understand the multiple functions of ALYREF and the CBC in RNA metabolism, we carried out structural and biochemical studies. We present cryo-EM structures of the CBC and CBC-ALYREF complexes at 3.38 Å and 3.22 Å resolution, respectively. The CBC-ALYREF structure reveals that both the NCBP1 and NCBP2 subunits of the CBC interact with ALYREF. HSV-1 ICP27 and HVS ORF57 target the binding interface between the RRM domain of ALYREF and the CBC. We suggest that these viruses not only hijack host pathways to export their own RNA but could also inhibit host RNA metabolism through their interactions with ALYREF. Structural overlay of CBC-ALYREF and EJC-ALYREF reveals that both the CBC and the EJC bind to the RRM domain of ALYREF in a mutually exclusive manner. This suggests that ALYREF’s interaction with the EJC is favored after ALYREF dissociates from the CBC, or as an independent event.

Results and discussion

ALYREF directly interacts with the CBC

The recombinant human ALYREF protein was shown to interact with the CBC in RNase-treated nuclear extracts (Cheng et al., 2006). Here, we used purified recombinant proteins to further investigate the molecular interactions between ALYREF and the CBC (Figure 1). It is well known that RS domain-containing proteins, including ALYREF, exhibit low solubility and are prone to aggregation. The addition of glutamic acid and arginine to the buffer can increase protein solubility and stability (Golovanov et al., 2004). Indeed, with a 1:1 mixture of glutamic acid and arginine, we were able to purify a GST-tagged ALYREF (residues 1–183) protein containing the N-terminal region and the RRM domain of ALYREF (Figure 1A). This construct includes the WxHD motif (residues 87–90), whose mutation was shown to affect interaction with the CBC in an immunoprecipitation study (Gromadzka et al., 2016). GST pull-down assays showed that ALYREF directly interacts with the CBC (Figure 1B).

Figure 1. 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). Protein constructs used in GST pull-down assays in panel (B) (NCBP1, residues 1–790; NCBP2, residues 1–156; ALYREF, residues 1–183) are indicated by black lines under the respective proteins. (B) ALYREF directly interacts with the CBC. In vitro GST pull-down assays were performed with purified recombinant human CBC and GST-tagged ALYREF or the corresponding construct of mouse ALYREF2 (mALYREF2, residues 1–155). Results are representative of three technical repeats.

Figure 1—source data 1. Original file for the gels in Figure 1B.

Figure 1.

Figure 1—figure supplement 1. Human ALYREF and mouse ALYREF2 are conserved.

Figure 1—figure supplement 1.

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.
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We next attempted to characterize the CBC-ALYREF interaction using cryo-EM. We found that human ALYREF is prone to aggregation in the absence of a GST tag. To obtain an untagged ALYREF protein amenable for structural studies, we tested the solubility of several ALYREF orthologs and found that mouse ALYREF2 (mALYREF2, residues 1–155) exhibited better solubility than the ALYREF construct utilized above. The conserved UBM motifs, the WxHD motif, and the RRM domain are nearly identical between ALYREF and mALYREF2 (Figure 1—figure supplement 1). Indeed, like ALYREF, mALYREF2 directly interacts with the CBC (Figure 1B). As the ALYREF/mALYREF2 interaction with the CBC is conserved and mALYREF2 exhibits better solubility, we focused on mALYREF2 in the cryo-EM investigations.

Cryo-EM structures of the CBC and CBC-ALYREF complexes

We collected cryo-EM data from the mixture of the CBC complex and mALYREF2 in the presence of the cap analog m7GpppG. The data yielded two maps that correspond to the CBC complex and the CBC-mALYREF2 complex, respectively. The CBC map was refined to an overall resolution of 3.38 Å (Figure 2A, Figure 2—figure supplement 1–3, Table 1). The electron density of the cap analog is clearly visible bound to NCBP2 (Figure 2—figure supplement 3C). Compared to unliganded CBC, the cap analog induces significant rearrangements in both the N-terminal extension and the C-terminal tail of NCBP2 (Figure 2B) to form critical interactions. For example, the N-terminal extension (residues 16–28) swings toward the central globular domain of NCBP2 and positions the Y20 residue to sandwich the cap analog with Y43 of NCBP2. These conformational changes were also observed in the crystal structures of ligand-bound CBC (Calero et al., 2002; Mazza et al., 2002). Overall, the cryo-EM structure of the CBC determined here resembles the previously reported crystal structures of the liganded CBC, with root mean squared deviation (RMSD) of 0.81 Å and 0.71 Å for NCBP1 and NCBP2, respectively (Calero et al., 2002).

Figure 2. 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). The 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.

Figure 2.

Figure 2—figure supplement 1. Workflow for cryo-EM data processing.

Figure 2—figure supplement 1.

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 3.38 Å resolution.
Figure 2—figure supplement 2. Cryo-EM reconstruction of the CBC.

Figure 2—figure supplement 2.

(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 of the CBC computed in CryoSPARC. (D) 3DFSC analysis of the CBC reconstruction. The sphericity and global resolution values are noted within the graph window. (E) Cryo-EM map of the CBC complex. NCBP1 and NCBP2 are colored in yellow and teal, respectively. (F) Local resolution of the CBC cryo-EM map.
Figure 2—figure supplement 3. Structural model of the CBC.

Figure 2—figure supplement 3.

(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 interface between NCBP1 and NCBP2 (B) and near m7G (C).
Figure 2—figure supplement 4. Cryo-EM reconstruction of the CBC-mALYREF2 complex.

Figure 2—figure supplement 4.

(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 distribution of the final reconstruction of CBC-mALYREF2 computed in CryoSPARC. (D) 3DFSC analysis of the CBC-mALYREF2 reconstruction. The sphericity and global resolution values are noted within the graph window. (E) Cryo-EM map of the CBC-mALYREF2 complex. NCBP1, NCBP2, and mALYREF2 are colored in yellow, teal, and red, respectively. (F) Local resolution of the CBC-mALYREF2 cryo-EM map.
Figure 2—figure supplement 5. Structural model of the CBC-mALYREF2 complex.

Figure 2—figure supplement 5.

(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 model of CBC-mALYREF2 at the interface between NCBP1 and NCBP2 (B) and near m7G (C). (D) Comparison of the CBC-mALYREF2 (colored as in Figure 2C) and the CBC (gray) cryo-EM structures. A loop (residues 38–45) of NCBP1, indicated by the arrow, becomes ordered upon mALYREF2 binding.
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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
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The CBC-mALYREF2 map was refined to an overall resolution of 3.22 Å (Figure 2C, Figure 2—figure supplements 1, 4 and Figure 2—figure supplement 5, Table 1). The cap analog is bound to NCBP2 within the CBC-mALYREF2 complex (Figure 2—figure supplement 5C). The structure shows that the RRM domain of mALYREF2 binds to both NCBP1 and NCBP2 subunits (Figure 2C and D). The N-terminal region of mALYREF2 (residues 1–73) does not show traceable density and is possibly disordered. mALRYEF binding does not induce a significant overall conformational change in CBC (Figure 2D, Figure 2—figure supplement 5D). Comparing CBC and CBC-mALYREF2, NCBP1 and NCBP2 have an RMSD of 0.32 Å and 0.30 Å, respectively. Locally, an NCBP1 loop in proximity to mALYREF2, formed by residues 38–45, becomes more ordered.

CBC-ALYREF interfaces

The interfaces between mALYREF2 and the CBC involve 16, 18, and 6 residues of mALYREF2, NCBP1, and NCBP2, respectively (Figure 3). The RRM domain of mALYREF2 assumes a canonical β1α1β2β3α2β4 topology (Figure 3A), forming an α-helical surface and a β-sheet surface. The α-helical surface recognizes the CBC through extensive hydrophilic and hydrophobic interactions (Figure 3B, Figure 3—figure supplement 1A and B). The α1 helix of mALYREF2 is enriched with acidic residues and makes key hydrophilic interactions with NCBP1. For example, E97 forms salt bridges with K330 and K381 of NCBP1. Y135 on the α2 helix of mALYREF2 makes a hydrogen bond with K330 of NCBP1. In addition, the loop between α2 and β4 of mALYREF2 forms hydrophobic interactions with NCBP1. V138, P139, and L140 of mALYREF2 bind to a hydrophobic pocket on NCBP1 formed by A334, V337, and L382.

Figure 3. 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 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. The surface of mALYREF2 is colored according to the electrostatic potential, ranging from red (–5 kBT/e) to blue (+5 kBT/e). Dotted line indicates a putative RNA binding path.

Figure 3.

Figure 3—figure supplement 1. Electron density maps at the CBC-mALYREF2 interfaces.

Figure 3—figure supplement 1.

Electron density map and structural model of CBC-mALYREF2 are shown in the same views as in Figure 3B, left panel (A), Figure 3B, right panel (B), and Figure 3C (C).
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The interface between mALYREF2 and NCBP2 is near the m7G binding pocket (Figure 3C, Figure 3—figure supplement 1C). The α2 helix of mALYREF2 contacts S13 and Y14 in the N-terminal extension of NCBP2. S13 and Y14 also directly interact with NCBP1 and are thought to enable the hinged motion of the N-terminal extension (residues 16–28) upon binding to the cap (Mazza et al., 2002). In addition, the α1 helix of mALYREF2 is in proximity to the R105 and I110 residues of NCBP2. NCBP2 exhibits a positively charged groove extending from the cap binding site, which is suggested to be an RNA binding site (Calero et al., 2002). Upon mALYREF2 binding, this groove is buried. Interestingly, mALYREF2 features a positively charged surface near the m7G site (Figure 3D). Conceivably, this positively charged surface on mALYREF2 could serve as an RNA binding site for the nucleotides following the cap.

Based on the CBC-mALYREF2 structure, we generated mutations (mut-1 and mut-2) on the RRM domain of human ALYREF (ALYREF-RRM, residues 103–183) to validate its interaction with the CBC. For ALYREF-RRM-mut-1 (Y166R/V169R/P170R), mutated residues are localized on the α2-β4 loop and correspond to residues Y135/V138/P139 in mALYREF2 (Figure 3A and B). For ALYREF-RRM-mut-2 (E124R/E128R), mutated residues are localized on the α1 helix and correspond to residues E93/E97 in mALYREF2 (Figure 3A and B). In agreement with the CBC-ALYREF structure, we found that the RRM domain of ALYREF directly interacts with the CBC, albeit with weaker interaction compared to ALYREF (residues 1–183) (Figures 1B and 4A). The difference likely results from the WxHD motif (residues 87–90) localized in the N-terminal region of ALYREF. Evidence suggests that mutation of the WxHD motif reduces ALYREF’s interaction with the CBC (Gromadzka et al., 2016). The WxHD motif may represent a second binding site for the CBC that remains to be characterized. Importantly, compared to the wild type protein, both ALYREF-mut-1 and mut-2 show reduced binding to the CBC (Figure 4A). Together, the mutagenesis studies validate the CBC-ALYREF interfaces observed in the structure.

Figure 4. Dissection of the ALYREF and CBC interfaces.

Figure 4.

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(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 technical repeats.

Figure 4—source data 1. Original file for the gels in Figure 4A.
Figure 4—source data 2. Original file for the gels in Figure 4B.

The CBC-mALYREF2 structure reveals that the interaction between ALYREF and the CBC mainly involves the NCBP1 subunit (Figure 3B and C). We further dissected the interaction between ALYREF and individual NCBP1 and NCBP2 subunits using GST pull-down assays. NCBP1 can be efficiently pulled down by GST-ALYREF, whereas NCBP2 did not show detectable interaction (Figure 4B). These results are consistent with the structural observations and indicate that NCBP1 is the major subunit of the CBC to interact with ALYREF.

CBC-ALYREF and 5′ cap-dependent mRNP export

ALYREF recruits the mRNP export machinery TREX complex to the 5′ end of mRNA through direct interactions with both the CBC and TREX (Cheng et al., 2006; Ren et al., 2017). The UBMs of ALYREF directly interact with the DDX39B component of the TREX complex (Ren et al., 2017). The N-terminal UBM is included in the ALYREF construct used for our cryo-EM studies but did not show visible electron density. Thus, this UBM is likely exposed and available to interact with DDX39B, which further connects to the entire TREX complex (Figure 5A). Consistently, ALYREF, DDX39B, THOC1, and THOC2 are present in NCBP1 immunoprecipitations from RNase-treated HeLa cell nuclear extracts (Cheng et al., 2006). In yeast, mutually exclusive interactions were shown between Yra1 with Sub2 and the NXF1-NXT1 ortholog Mex67-Mtr2 (Strässer et al., 2002). So, the ALYREF-dependent NXF1-NXT1 loading on mRNA likely occurs after DDX39B dissociates from ALYREF. The CBC could also function as a landing pad for ALYREF as previously proposed (Viphakone et al., 2019). After recruitment to the 5′ end of mRNA by the CBC, ALYREF could then transfer away from the 5′ end, to other sites enriched with export factors and participate in different complexes located along the mRNA. In addition to the ALYREF-NXF1-NXT1 complex, some other ALYREF containing complexes could exist on the same mRNP, such as the complex of ALYREF/DDX39B/SARNP, which facilitates high-order mRNP assembly (Dufu et al., 2010; Xie et al., 2023).

Figure 5. 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) Proposed 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 the nuclear export of viral mRNAs.

Figure 5.

Figure 5—figure supplement 1. HSV-1 ICP27 binds to the RRM domain of ALYREF and interferes with the CBC-ALYREF interaction.

Figure 5—figure supplement 1.

(A) NMR structure of the ALYREF-ICP27 complex (PDB ID 2KT5). (B) The CBC-ALYREF structure is overlayed with the ALYREF-ICP27 structure.
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The process of mRNP export, with the 5′ end exiting first from the NPC, has been shown in both insect and human systems using electron microscopy and single-molecule imaging techniques (Mehlin et al., 1992; Ashkenazy-Titelman et al., 2022). Interestingly, in the latter study, several adjacent NPCs were found to engage in the export of the same mRNA (Blobel, 1985). This observation is reminiscent of the gene gating hypothesis, which suggested that transcriptionally active genes are physically tethered to the site of mRNA export at the NPC (Blobel, 1985). Gated genes have been shown in yeast, worms, flies, and humans (Burns and Wente, 2014; Scholz et al., 2019). For these gated genes, the 5′ directionality of mRNA export could be primarily driven by the key placement of crucial RNA export factors at the 5′ end of the gene as illustrated here (Figure 5A), and this localization of export factors could greatly increase the efficiency of co-transcriptional processing and export.

CBC-ALYREF and viral hijacking of host mRNA export pathway

HSV-1 ICP27 and HVS ORF57 hijack the host mRNA export pathway through interactions with ALYREF. The RRM domain of ALYREF is targeted by both HSV-1 ICP27 and HVS ORF57 with overlapping interfaces (Figure 5B, Figure 5—figure supplement 1). Structural comparison between CBC-ALYREF, ALYREF-ICP27, and ALYREF-ORF57 reveals that the interface between ALYREF’s RRM domain with the CBC is not compatible with the ICP27/ORF57-ALYREF interactions (Figure 5B, Figure 5—figure supplement 1). In addition, in vivo data show that the ORF57 ortholog from Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) can still form a complex with ALYREF and the CBC (Boyne et al., 2008). So, it is likely that although ALYREF’s RRM domain interface with the CBC could be disrupted by ORF57, ALYREF can still use the WxHD motif to interact with the CBC (Figure 5C). Using this strategy, a virus can hijack the host pathway while simultaneously disrupting host interactions and processes. It should also be noted that the CBC, ALYREF, ICP27, and ORF57 are all RNA binding proteins. In addition to the protein-mediated interactions discussed above, RNA interactions should be considered, especially under the in vivo setting (Figure 5C). NXF1-NXT1 and DDX39B, the cellular ALYREF interacting proteins, are also hijacked by other factors from viruses. NXF1-NXT1 is targeted by influenza A virus NS1 protein (Zhang et al., 2019b) and SARS-CoV-2 Nsp1 protein (Zhang et al., 2021; Mei et al., 2024). DDX39B is targeted by influenza A virus NP protein (Momose et al., 2001; Morris et al., 2020). The molecular mechanisms revealed here and from previous studies pave the way for new useful targets in antiviral therapeutics.

Functional interplay of CBC-ALYREF and mRNP export factors

Transcription, splicing, and export are all tightly linked processes. The CBC promotes splicing through its binding partners, such as SRSF1 (Lenasi et al., 2011; Pabis et al., 2013). The detailed molecular interaction between the CBC and SRSF1 is revealed in a human pre-Bact-1 spliceosome structure (Townsend et al., 2020). The NCBP2 subunit of the CBC is the major binding site for SRSF1. Structural overlay of this structure with the CBC-ALYREF structure shows no significant steric hindrance between SRSF1 and ALYREF (Figure 6A). However, whether the CBC-ALYREF-SRSF1 complex exits in vivo and how their functions might be coordinated require further studies. After splicing, SRSF1 is mainly deposited on exons (Das and Krainer, 2014; Pandit et al., 2013). SRSF1 functions in mRNA export through interaction with the export receptor NXF1-NXT1 (Huang et al., 2004; Reed and Cheng, 2005; Müller-McNicoll et al., 2016). Of note, the interaction between both ALYREF and SR proteins with NXF1-NXT1 is regulated by phosphorylation. Only hypophosphorylated SR proteins can bind to NXF1-NXT1 efficiently (Huang et al., 2004; Okada et al., 2008; Xie et al., 2021b). Interestingly, SR proteins, such as Gbp2 and Hrb1 in Saccharomyces cerevisiae, also interact directly with the TREX complex (Xie et al., 2021a; Hurt et al., 2004), suggesting coordinated actions of TREX and SR proteins in mRNA export.

Figure 6. ALYREF and CBC in splicing and export.

Figure 6.

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(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) Proposed model of the mRNP export receptor NXF1-NXT1 recruitment by CBC-ALYREF and other factors during mRNA maturation.

Unlike the CBC and SRSF1 interfaces discussed above, the interaction between the ALYREF RRM domain and the CBC is incompatible with the interface between the ALYREF RRM domain and the EJC subunit MAGOH (Pacheco-Fiallos et al., 2023; Figure 6B). Mutation of the ALYREF WxHD motif affects its interaction with both the CBC and the EJC subunit eIF4A3 (Gromadzka et al., 2016). As both the WxHD motif and the RRM domain of ALYREF are mutually exclusive binding sites for the CBC and the EJC, the formation of the EJC-ALYREF complex likely happens after ALYREF dissociates from the CBC. It is also possible that the EJC-ALYREF interaction is independent of the CBC (Figure 6C). This possibility is supported by the report that ALYREF can be recruited to RNA by both CBC-dependent and -independent mechanisms (Nojima et al., 2007). The resulting mRNP with multiple copies of ALYREF and NXF1-NXT1, each recruited through different mechanisms and at different sites on the mRNP (Figure 6C), could exhibit increased export efficiency.

Conclusion and perspectives

The CBC plays important roles in multiple steps of mRNA metabolism through interactions with a plethora of factors. Here, we present the structural basis of the interaction between the CBC and a key mRNA export factor, ALYREF. The CBC-ALYREF structure reveals molecular insights into the ALYREF-mediated recruitment of mRNA export machinery to the 5′ end of nascent transcripts. We suggest working models for the coordinated events mediated by the CBC and ALYREF during splicing and mRNA export. Notably, both the CBC and ALYREF have been implicated in cancer. Mutations of the CBC residues interfacing with ALYREF, including K330N of NCBP1, R105C of NCBP2, and I110M of NCBP2, are found in several forms of cancers (Tate et al., 2019; Heath et al., 2021; Zhang et al., 2019a). These mutations could reduce ALYREF interaction and subsequently cause dysregulation of mRNA export and the processes that are coordinated by CBC and ALYREF. In addition, ALYREF has been shown to be frequently upregulated in various cancerous tissues (Domínguez-Sánchez et al., 2011). Determining whether dysregulation of the CBC-ALYREF interaction contributes to cancer pathogenesis, including characterization of the impact of cancer-associated mutations of the CBC, is an interesting area for future studies.

To date, it is unclear whether the functional connection between the CBC and ALYREF is conserved in yeast. In humans, recruitment of the mRNA export machinery is mediated by ALYREF in a CBC and EJC-dependent manner (Cheng et al., 2006; Gromadzka et al., 2016). Our work and studies by others (Pacheco-Fiallos et al., 2023; Gromadzka et al., 2016) reveal that both the CBC and the EJC recognize ALYREF through the WxHD motif and the RRM domain of ALYREF. There are notable differences between yeast and humans regarding ALYREF function: (1) the EJC is not present in the budding yeast, S. cerevisiae, and (2) the yeast ortholog of ALYREF, Yra1, does not contain a WxHD motif. Nevertheless, a study conducting transcriptome-wide mapping of mRNA biogenesis factors showed that Yra1 is enriched at the 5′ region of the mRNA (Baejen et al., 2014), raising the possibility that Yra1 localization could be mediated by the CBC. Further studies are required to reveal the mechanism of Yra1 in the context of the CBC and recruitment of the mRNA export machinery in yeast.

Materials and methods

Plasmids

Human NCBP1 (UniProt Q09161) was cloned into the pFastBac HTc vector with an N-terminal TEV cleavable His tag. Human NCBP2 (UniProt P52298) was cloned into a modified pFastBac1 vector containing an N-terminal TEV cleavable GST tag. Human ALYREF (UniProt Q86V81) and mouse ALYREF2 (UniProt Q4KL64) constructs were cloned into a modified pGST-4T-1 vector containing an N-terminal TEV cleavable GST tag.

Protein expression and purification

The NCBP1-NCBP2 complex was expressed in High-Five insect cells (Invitrogen) by coinfection of recombinant baculoviruses. Individual NCBP1 and NCBP2 subunits were expressed in High-Five insect cells infected with the respective recombinant baculovirus. High-Five cells were harvested 48 hr after infection and lysed in a buffer containing 50 mM Tris pH 8.0, 300 mM NaCl, 0.2 mM AEBSF, 2 mg/L aprotinin, 1 mg/L pepstatin, 1 mg/L leupeptin, and 0.5 mM TCEP. Proteins were purified using Glutathione Sepharose 4B resin (Cytiva) for NCBP2 and NCBP1-NCBP2, or Ni Sepharose 6FF resin (Cytiva) for NCBP1 alone. The proteins were further purified on a mono Q column (Cytiva). The expression tags were removed by overnight incubation with GST-TEV (for NCBP2 and NCBP1-NCBP2) or His-TEV (for NCBP1) at 4°C. The samples were passed through Glutathione Sepharose 4B resin (for NCBP2 and NCBP1-NCBP2) or Ni Sepharose 6FF resin (for NCBP1) to remove undigested protein and TEV. The proteins were further purified on a Superdex 200 column (Cytiva) in 10 mM Tris pH 8.0, 300 mM NaCl, and 0.5 mM TCEP.

GST-tagged ALYREF (residues 1–183), ALYREF-RRM (residues 103–183), ALYREF-RRM-mut-1 (Y166R/V169R/P170R), ALYREF-RRM-mut-2 (E124R/E128R), and mALYREF2 (residues 1–155) were expressed in Escherichia coli Rosetta cells (Sigma-Aldrich). Protein expression was induced with 0.5 mM IPTG at 16°C overnight. Cells were lysed in a buffer containing 50 mM Tris, pH 8.0, 500 mM NaCl, 50 mM glutamic acid, 50 mM arginine, 0.5 mM TCEP, 0.2 mM AEBSF, and 2 mg/L aprotinin. The GST-tagged proteins were pulled down using Glutathione Sepharose 4B resin. GST-tagged ALYREF-RRM wild type and mutant proteins were purified on a Superdex 200 column in 10 mM Tris pH 8.0, 300 mM NaCl, and 5 mM DTT. GST-tagged ALYREF (residues 1–183) was purified by a cation exchange column (source 15S, Cytiva), followed by a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 500 mM NaCl, 50 mM glutamic acid, 50 mM arginine, and 0.5 mM TCEP. GST-tagged mALYREF2 (residues 1–155) was purified by an anion exchange column (Q Sepharose, Cytiva), followed by a cation exchange column (SP Sepharose, Cytiva). For untagged mALYREF2 used in cryo-EM studies, the GST tag was removed by overnight incubation with GST-TEV at 4°C. Untagged mALYREF2 was further purified on a cation exchange HiTrap SP column (Cytiva), followed by a Superdex 200 column equilibrated with 10 mM Tris, pH 8.0, 150 mM NaCl, 50 mM glutamic acid, 50 mM arginine, and 0.5 mM TCEP.

All purified proteins were flash frozen in liquid nitrogen, and stored at –80 °C.

Cryo-EM sample preparation and data collection

NCBP1-NCBP2 at 1.2 μM was incubated with mALYREF2 (residues 1–155) at 3.6 μM in the presence of the 5′ cap analog m7GpppG (NEB) at 500 μM at 4°C for 0.5 hr. The sample was deposited on glow-discharged UltrAuFoil R 1.2/1.3 grids (Quantifoil). Grids were blotted for 6 s with a blotting force of 6 at 4°C and 100% humidity and plunged into liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher). The data were collected with a Glacios Cryo-TEM (Thermo Fisher) equipped with a Falcon 4i detector (Thermo Fisher). Movies were collected with EPU at a magnification of 190,000×, corresponding to a calibrated pixel size of 0.732 Å/pixel. A total of 5858 movies recorded in EER format were collected with a defocus range from 1.0 μm to 2.0 μm. A full description of the cryo-EM data collection parameters can be found in Table 1.

Cryo-EM image processing and model building

Cryo-EM data were processed with cryoSPARC (Punjani et al., 2017). Movies in EER format were gain normalized, aligned, and dose-weighted using patch motion correction, followed by patch CTF estimation. A subset of 100 micrographs was subjected to blob particle picking followed by 2D classification to obtain particles for Topaz training. 1,625,826 particles were picked by Topaz from the entire dataset and were extracted with a box size of 288 × 288 pixels. 1,453,188 particles were retained after 2D classification. The particles were subjected to ab initio model reconstruction and heterogeneous refinement, resulting in one good class from 637,185 particles, which showed robust CBC densities as well as densities corresponding to mALYREF2 along with ‘junk’ classes. Further heterogeneous refinement resulted in a set of 366,410 particles corresponding to the CBC-mALREF2 complex and a set of 121,056 particles corresponding to the CBC complex. The CBC-mALYREF2 particles were subjected to another round of heterogeneous refinement and homogeneous refinement, which yielded a reconstruction from 241,915 particles with an overall resolution of 3.22 Å. Directional anisotropy of the CBC-mALYREF2 map was assessed using the 3DFSC server (Tan et al., 2017; https://3dfsc.salk.edu), which indicates a sphericity of 0.946. The CBC particles were subjected to another round of heterogeneous refinement, homogeneous refinement, and non-uniform refinement, which yielded a reconstruction from 78,039 particles with an overall resolution of 3.38 Å. 3DFSC analysis of the CBC map indicates a sphericity of 0.962.

An initial model of CBC was obtained by docking AlphaFold models of NCBP1 (AF-Q09161-F1) and NCBP2 (AF-P52298-F1). An initial model of CBC-mALYREF2 was obtained by docking AlphaFold models of NCBP1, NCBP2, and mALYREF2 into the cryo-EM density map. The models were adjusted in Coot (Emsley et al., 2010), followed by real-space refinement in Phenix (Afonine et al., 2012). The final CBC model contains NCBP1, NCBP2, and the m7G moiety of m7GpppG. The final CBC-mALYREF2 model contains NCBP1, NCBP2, the RRM domain of mALYREF2, and the m7G moiety of m7GpppG. Figures were prepared using PyMOL (Molecular Graphics System, Schrodinger, LLC) and Chimera (Luo et al., 2001).

GST pull-down

GST or GST-tagged proteins were pre-incubated with GST resin in binding buffer (10 mM Tris pH 8.0, 500 mM NaCl, 50 mM glutamic acid, 50 mM arginine, 0.5 mM TCEP) on ice for 0.5 hr and were mixed with gentle tapping every 3–5 min. The beads were then washed three times with 600 μl of buffer containing 10 mM Tris pH 8.0, 100 mM NaCl (Figures 1B and 4B) or 50 mM NaCl (Figure 4A), and 0.5 mM TCEP. NCBP1, NCPB2, or the NCBP1-NCBP2 complex was incubated with the 5′ cap analog m7GpppG at 20 μM, adjusted salt concentration to 100 mM NaCl (Figures 1B and 4B) or 50 mM NaCl (Figure 4A), and then added to beads. The samples were incubated on ice for 0.5 hr and mixed with gentle tapping every 3–5 min. Beads were then washed three times with 600 μl of wash buffer (10 mM Tris pH 8.0, 50 mM NaCl, 0.5 mM TCEP) before bound proteins were eluted in 10 mM Tris pH 8.0, 500 mM NaCl (Figures 1B and 4B) or 150 mM NaCl (Figure 4A), 25 mM glutathione, and 0.5 mM TCEP. 6% (Figures 1B and 4B) or 3% (Figure 4A) of the input and 60% of the eluted proteins were analyzed using Coomassie stained SDS-PAGE gels. The experiments were repeated three times independently.

Acknowledgements

We thank Melissa Chambers, Scott Collier, and Mariam Haider at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University for assistance in Cryo-EM data collection. We acknowledge the use of the Glacios cryo-TEM, which was acquired by NIH grant S10 OD030292-01. This work was supported by NIH R35 GM133743 (YR) and R01 AI184975 (YR). BPC was in part supported by NIH/NCI training grant T32 CA119925.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Yihu Xie, Email: yihu.xie@vanderbilt.edu.

Yi Ren, Email: yi.ren@vanderbilt.edu.

Yang Yang, Iowa State University, United States.

Amy H Andreotti, Iowa State University, United States.

Funding Information

This paper was supported by the following grants:

  • National Institutes of Health R35 GM133743 to Bradley P Clarke, Alexia E Angelos, Menghan Mei, Pate S Hill, Yihu Xie, Yi Ren.

  • National Institutes of Health T32 CA119925 to Bradley P Clarke.

  • National Institutes of Health R01 AI184975 to Menghan Mei, Pate S Hill, Yihu Xie, Yi Ren.

Additional information

Competing interests

No competing interests declared.

Author contributions

Data curation, Formal analysis, Writing - original draft, Writing - review and editing.

Data curation, Formal analysis, Writing - review and editing.

Data curation, Formal analysis, Writing - review and editing.

Data curation.

Data curation, Formal analysis, Supervision, Writing - original draft, Writing - review and editing.

Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing.

Additional files

MDAR checklist

Data availability

The coordinates of CBC-ALYREF and CBC have been deposited in PDB under accession codes 8SRR and 8SUY. The corresponding density maps have been deposited in EMDB under accession codes EMD-40739 and EMD-40780. Source data files have been provided for Figures 1 and 4.

The following datasets were generated:

Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) RCSB Protein Data Bank. 8SUY

Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. RCSB Protein Data Bank. 8SRR

Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. Electron Microscopy Data Bank. EMD-40739

Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) Electron Microscopy Data Bank. EMD-40780

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eLife assessment

Yang Yang 1

This important study reports the cryo-electron microscopy structure of a multi-protein complex that recognizes the 5'-end cap of mRNAs and plays a critical role in mRNA export. The structural and biochemical analyses in this study provide convincing evidence to support the major claims of the authors, with the inclusion of more functional characterizations in cell-based systems having corroborated the claims further and thus strengthening the study. This paper would be of interest to structural biologists and RNA biologists working on mRNA metabolism.

Reviewer #1 (Public Review):

Anonymous

Summary:

The authors use a combination of biochemistry and cryo-EM studies to explore a complex between the cap binding complex and an RNA binding protein, ALYREF, that coordinates mRNA processing and export.

Strengths:

The biochemistry and structural biology are supported by mutagenesis that tests the model in vitro. The structure provides new insight into how key events in RNA processing and export are likely to be coordinated.

Weaknesses:

The authors provide biochemical studies to confirm the interactions that they identify; however, they do not perform any studies to test these models in cells or explore the consequences for mRNA export from the nucleus. In fact, several of the amino acids that they identified in ALYREF that are critical for the interaction, as determined by their own biochemical studies are conserved in budding yeast Yra1 (residues E124/E128 are E/Q in budding yeast and residues Y135/V138/P139 are F/S/P), where the impact on poly(A) RNA export from the nucleus could be readily evaluated. The authors mention the potential for future studies in the manuscript, but they do not perform any analysis in this study that would explore the contributions of these new interactions.

Reviewer #2 (Public Review):

Anonymous

Summary:

In this manuscript, Bradley and his colleagues represented the cryo-EM structure of the nuclear cap-binding complex (CBC) in complex with an mRNA export factor, ALYREF, providing a structural basis for understanding CBC regulating gene expression.

Strengths:

The authors successfully modeled the N-terminal region and the RRM domain of ALYREF (residues 1-183) within the CBC-ALYREF structure, which revealed that both the NCBP1 and NCBP2 subunits of the CBC interact with the RBM domain of ALYREF. Further mutagenesis and pull-down studies provided additional evidence to the observed CBC-ALYREF interface. Additionally, the authors engaged in a comprehensive discussion regarding other cellular complexes containing CBC and/or ALYREF components. They proposed potential models that elucidated coordinated events during mRNA maturation. This study provided structural evidence to show how CBC effectively recruits mRNA export factor machinery, enhancing our understanding of CBC regulating gene expression during mRNA transcription, splicing, and export.

Weaknesses:

Absence of functional data to support the proposed models in this study.

Reviewer #3 (Public Review):

Anonymous

Summary:

The authors carried out structural and biochemical studies to investigate the multiple functions of CBC and ALYREF in RNA metabolism.

Strengths:

For the structural study part, the authors successfully revealed how NCBP1 and NCBP2 subunits interact with mALYREF (residues 1-155). Their binding interface was then confirmed by biochemical assays (mutagenesis and pull-down assays) presented in this study.

Weaknesses:

The model derived from the cryo-EM structure will likely need to be validated in future functional studies.

eLife. 2024 Sep 16;12:RP91432. doi: 10.7554/eLife.91432.3.sa4

Author response

Bradley P Clarke 1, Alexia E Angelos 2, Menghan Mei 3, Pate S Hill 4, Yihu Xie 5, Yi Ren 6

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Reviews):

Summary:

The authors use a combination of biochemistry and cryo-EM studies to explore a complex between the cap-binding complex and an RNA binding protein, ALYREF, that coordinates mRNA processing and export.

Strengths:

The biochemistry and structural biology are supported by mutagenesis which tests the model in vitro. The structure provides new insight into how key events in RNA processing and export are likely to be coordinated.

Weaknesses:

The authors provide biochemical studies to confirm the interactions that they identify; however, they do not perform any studies to test these models in cells or explore the consequences of mRNA export from the nucleus. In fact, several of the amino acids that they identified in ALYREF that are critical for the interaction, as determined by their own biochemical studies, are conserved in budding yeast Yra1 (residues E124/E128 are E/Q in budding yeast and residues Y135/V138/P139 are F/S/P), where the impact on poly(A) RNA export from the nucleus could be readily evaluated. The authors could at least mention this point as part of the implications and the need for future studies. No one seems to have yet targeted any of these conserved residues, so this would be a logical extension of the current work.

We thank the reviewer for the feedback on our work. ALYREF coordinates pre-mRNA processing and export through interactions with a plethora of mRNA biogenesis factors including the DDX39B subunit of the TREX complex, CBC, EJC, and 3’ processing factors. ALYREF mediates the recruitment of the TREX complex on nascent transcripts which depends on its interactions with both CBC and EJC. Our work and studies by others indicate that ALYREF uses overlapping interfaces including both the N-terminal WxHD motif and the RRM domain to bind CBC and EJC. Thus, ALYREF mutants deficient in CBC interaction will also disrupt the ALYREF-EJC interaction and are not ideal for functional studies. In addition, the CBC plays important roles in multiple steps of mRNA metabolism through interactions with a plethora of factors, which often interact competitively with CBC. Identification of separation-of-function mutations on CBC or ALYREF that specifically disrupt their interaction but not other cellular complexes containing CBC or ALYREF would be an important future area to test the model in cells.

We appreciate the reviewer’s insightful comments regarding yeast Yra1. Thus far, the physical and functional connection between Yra1 and CBC in yeast has not been demonstrated. There are major differences between yeast Yra1 and human ALYREF. Given the lack of an EJC in S. cerevisiae, it is unclear whether Yra1 acts in a similar manner as human ALYREF. In addition, Yra1 does not contain a WxHD motif in its N-terminal unstructured region, which is involved in CBC and EJC interactions in ALYREF. Characterization of the Yra1-CBC interaction will be an interesting future direction. We now include a discussion about yeast Yra1 in the newly added “Conclusion and perspectives” section.

Specific suggestions:

The authors could put their work in context by speculating how some of the amino acids that they identify as being critical for the interactions they identify could contribute to cancer. For example, they mention mutations of interacting residues in NCBP2 are associated with human cancers, pointing out that NCBP2 R105C amino acid substitution has been reported in colorectal cancer and the NCBP2 I110M mutation has been found in head and neck cancer. Do the authors speculate that these changes would decrease the interaction between NCBP2 and ALYREF and, if so, how would this contribute to cancer? They also mention that a K330N mutation in NCBP1 in human uterine corpus endometrial carcinoma, where Y135 on the α2 helix of mALYREF2 makes a hydrogen bond with K330 of NCBP1. How do they speculate loss of this interaction would contribute to cancer?

In the revised manuscript, we include a discussion about these CBC mutants found in human cancers in the “Conclusion and perspectives” section. We think some of these CBC mutants, such as NCBP-1 K330N, could reduce interaction with ALYREF. Compromised CBC-ALYREF interaction will affect the recruitment of the TREX complex on nascent transcripts and cause dysregulation of mRNA export. In addition, that could also change the partition of CBC and ALYREF in different cellular complexes and cause perturbation of various steps in mRNA biogenesis that are regulated by CBC and ALYREF. Thus far, it is unclear whether and how loss of the CBC-ALYREF interaction directly contributes to cancer. Our work and that of others provide molecular insights to test in future studies.

Reviewer #2 (Public Reviews):

Summary:

In this manuscript, Bradley and his colleagues represented the cryo-EM structure of the nuclear cap-binding complex (CBC) in complex with an mRNA export factor, ALYREF, providing a structural basis for understanding CBC regulating gene expression.

Strengths:

The authors successfully modeled the N-terminal region and the RRM domain of ALYREF (residues 1-183) within the CBC-ALYREF structure, which revealed that both the NCBP1 and NCBP2 subunits of the CBC interact with the RBM domain of ALYREF. Further mutagenesis and pull-down studies provided additional evidence to the observed CBC-ALYREF interface. Additionally, the authors engaged in a comprehensive discussion regarding other cellular complexes containing CBC and/or ALYREF components. They proposed potential models that elucidated coordinated events during mRNA maturation. This study provided good evidence to show how CBC effectively recruits mRNA export factor machinery, enhancing our understanding of CBC regulating gene expression during mRNA transcription, splicing, and export.

Weaknesses:

No in vivo or in vitro functional data to validate and support the structural observations and the proposed models in this study. Cryo-EM data processing and structural representation need to be strengthened.

We appreciate the reviewer’s comments and suggestions. The fact that ALYREF uses highly overlapped binding interfaces for CBC and EJC interactions prevents us from a clear functional dissection of the ALYREF-CBC interaction using in vitro assays or in cells at the current stage. Please also see our response to Reviewer 1.

In this revised manuscript, we have reprocessed the cryo-EM data using a different strategy which yields significantly improved maps. We have made improvements to the presentation of the structural work based on the reviewer’s specific comments.

Reviewer #3 (Public Reviews):

Summary:

The authors carried out structural and biochemical studies to investigate the multiple functions of CBC and ALYREF in RNA metabolism.

Strengths:

For the structural study part, the authors successfully revealed how NCBP1 and NCBP2 subunits interact with mALYREF (residues 1-155). Their binding interface was then confirmed by biochemical assays (mutagenesis and pull-down assays) presented in this study.

Weaknesses:

The authors did not provide functional data to support their proposed models. The authors should include more details regarding the workflow of their cryo-EM data processing in the figure.

We thank the reviewer for the comments. We completely agree that testing the proposed models in cells would be ideal. However, as we also respond to the other reviewers, functional studies are premature at the current stage because both ALYREF and CBC are components of many cellular complexes that regulate mRNA metabolism. Separation-of-function mutations on CBC or ALYREF first need to be identified in future studies for further investigation. Please also see our response to Reviewer 1.

As suggested by the reviewer, we have included more details of the cryo-EM workflow in this revised manuscript. We have also included various validation measures including 3DFSC analyses, map vs model FSC curves, and representative density maps at various protein-protein binding interfaces.

Recommendations for the Authors:

Reviewer #1 (Recommendations for the Authors):

Major points:

The authors should take advantage of Figure 1, which shows the domain structures of NCBP1, NCBP2, and ALYREF to indicate for the reader specifically which protein domains are included in the biochemical and structural analyses. In the current version of the manuscript, there is plenty of space to indicate below each domain structure precisely what regions are included.

In this revised manuscript, we have revised Figure 1A to indicate the protein constructs used in this work.

Although it is fine to combine the Results and Discussion, the authors should really offer a concluding paragraph to highlight the novel results from this study and put the results in context.

We thank the reviewer for the recommendation. We now include a “Conclusion and perspectives” section in this revised manuscript.

Minor comments:

Page 5, last sentence (and others) starts a sentence with the word "Since" when likely "As" which does not imply a time element to the phrase, is the correct word.

"Since the ALYREF/mALYREF2 interaction with the CBC is conserved and mALYREF2 exhibits better solubility, we focused on mALYREF2 in the cryo-EM investigations."

Would be more correct as: "As the ALYREF/mALYREF2 interaction with the CBC is conserved and mALYREF2 exhibits better solubility, we focused on mALYREF2 in the cryo-EM investigations."

We thank the reviewer for the comments. We have made the corrections.

The word 'data' is plural so the sentence at the bottom of p.9 that includes the phrase "...in vivo data shows.." should read "..in vivo data show.."

Corrected in the revised manuscript.

Reviewer #2 (Recommendations for the Authors):

Major points:

(1) The authors claimed the improved solubility of mouse ALYREF2 (mALYREF2, residues 1-155) compared to the previously employed ALYREF construct. However, human ALYREF has already been purified successfully for pull down assay, indicating soluble human ALYREF obtained, why not use human ALYREF directly? Please clarify.

Pull-down studies were performed with GST-tagged ALYREF. For cryo-EM studies, untagged ALYREF is preferred to avoid potential issues that may arise from the expression tag. However, untagged ALYREF is less soluble than GST-tagged ALYREF and is not amenable for structural studies. We have revised the text to clarify this point.

(2) The authors confirmed CBC-ALYREF interfaces through mutagenesis and pull-down assays in vitro. However, it would be more informative and interesting to include functional assays in vitro or/and in vivo with mutagenesis.

We completely concur with the reviewer that testing the proposed models in vitro and in vivo would be important. However, as we pointed out in our response to public reviews, the highly overlapped binding interfaces on ALYREF for CBC and EJC interactions pose a great challenge for functional studies. Furthermore, both ALYREF and CBC are multifunctional factors and interact with a number of partners. Ideally, separation-of-function mutants that specifically disrupt the CBC-ALYREF interaction but not others need to be identified in future studies in order to perform functional studies.

(3) About cryo-EM data processing and structural representation:

(1) In the description of the cryo-EM data processing, the authors claimed they did heterogeneous refinement, homogenous refinement, and then local refinement. This reviewer is puzzled by this process because the normal procedure should be non-uniform refinement following homogenous refinement. If the authors did not perform non-uniform refinement, they should do it because it would significantly improve the quality and resolution of cryo-EM maps. In addition, the right local refinement should include mask files and only show the density/map of the local region.

We thank the reviewer for the suggestions. In response to the reviewer’s comment on the preferred orientation issue (point 5 below), we reprocessed the cryo-EM data and obtained significantly improved cryo-EM maps. In this revised manuscript, the CBC-mALYREF map was refined using homogeneous refinement; the CBC map was refined using homogenous refinement followed by non-uniform refinement. Refinement masks are included in Figure 2-figure supplement1.

(2) Further local refinements with signal subtraction should be performed to improve the density and resolution of mALYREF2.

We tested local refinement with or without signal subtraction using masks covering mALYREF2 and various regions of CBC. Unfortunately, this approach did not improve the density of mALYREF2. We suspect that the small size of mALYREF2 (77 residues for the RRM domain) and the intrinsic flexibility of CBC are the limiting factors in these attempts.

(3) Figures with cryoEM map showing the side chains of the residues on the CBC-mALYREF2 interface should be included to strengthen the claims. Authors could add the map to Figure 3b/c or present it as a supplementary figure.

We include new supplementary figures (Figure 3-figure supplement 1) to show the electron densities corresponding to the views in Figure 3B and 3C. Residues labeled in Figure 3B and 3C are shown in sticks in these supplementary figures.

(4) For cryo-EM date processing, the authors have omitted lots of important details. Could the authors elaborate on the data processing with more details in the corresponding Figure and Methods Sections? Only one abi-initial model from the picked good particles was displayed in the figure. Are there any other different conformations of 3D classes for the dataset? In addition, too few classes have been considered in 3D classification, more classes may give a class with better density and resolution.

We thank the reviewer for the comments. We have reprocessed the cryo-EM data. A major change is to use Topaz for particle picking. We now include more details for data processing in Figure 2-figure supplement 1 and the method section. The cryo-EM sample is relatively uniform. Ab-initio reconstruction and heterogenous refinement yielded only one good class and the other classes are “junk” classes (omitted in the workflow figure). No major conformational changes were observed throughout the multiple rounds of heterogenous refinement for both CBC and CBCmALYREF2. In this revised manuscript, we have been able to obtain significantly improved maps through the new data processing strategy employing Topaz as illustrated in Figure 2-figure supplement 1 to 5.

(5) Angular distribution plots should be included to show if there is a preferred orientation issue. Based on the presented maps in validation reports, there may exist a preferred orientation issue for the reported two cryo-EM maps. Detailed 3D-Histogram and directional FSC plots for all the cryo-EM maps using 3DFSC web server should be presented to show the overall qualities (https://www.nature.com/articles/nmeth.4347 and https://3dfsc.salk.edu/).

We thank the reviewer for the recommendations. In response to the reviewer’s comment on the preferred orientation issue, we reprocessed the cryo-EM data. Topaz was used for particle picking instead of template picking. 3DFSC analyses indicate that the new CBC-mALREF2 map has a sphericity of 0.946, which is a significant improvement from the previous map which has a sphericity of 0.815. Consistently, the maps presented in this revised manuscript show significantly improved densities. We now include angular distribution and 3DFSC analyses of the EM maps (Figure 2-figure supplement 2 and 4).

(6) Figures of model-to-map FSCs need to be present to demonstrate the quality of the models and the corresponding ones (model resolution when FSC=0.5) should also be included in Table 1. The accuracy of the model is important for structural explanations and description.

The model-to-map FSCs are now included in Figure 2-figure supplement 3A and 5A. The model resolutions of CBC-mALYREF2 and CBC are estimated to be 3.5 Å and 3.6 Å at an FSC of 0.5. These numbers are now included in Table 1.

(7) In addition, figures of local density maps with different regions of the models, showing side chains, are necessary and important to justify the claimed resolutions.

We now include density maps overlayed with residue side chains at various regions. For the CBCmALYREF2 map, density maps are shown at the mALYREF2-NCBP1 interfaces (Figure 3-figure supplement 1A and 1B), mALYREF2-NCBP2 interface (Figure 3-figure supplement 1C), NCBP1NCPB2 interface (Figure 2-figure supplement 5B), and the region near m7G (Figure 2-figure supplement 5C). For the CBC map, density maps are shown at the NCBP1-NCPB2 interface (Figure 2-figure supplement 3B) and the region near m7G (Figure 2-figure supplement 3C).

Minor points:

(1) A figure superimposing the models from the CBC-mALYREF2 amp and mALYREF2 alone map is necessary to present that there are no other CBC binding-induced conformational changes in CBC except the claimed by the authors. In addition, a figure showing the density of m7GpppG should be included as well.

Overlay of CBC and CBC-mALYREF2 models is now presented in Figure 2-figure supplement 3D. Comparing CBC and CBC-mALYREF2, NCBP1 and NCBP2 have a RMSD of 0.32 Å and 0.30 Å, respectively. The density maps near the M7G cap analog are shown in Figure 2-figure supplement 3C for CBC and Figure 2-figure supplement 5C for CBC-mALYREF2.

(2) Authors obtained the two maps from one dataset, so "we first determined" and "we next determined" (page 6) should be replaced with something like "One class of 3D cryo-EM map revealed' and "Another class of 3D cryo-EM map defined".

We have revised the text as suggested by the reviewer.

(3) In 'Abstract', 'a mRNA export factor' should be 'an mRNA export factor'.

Corrected in the revised manuscript.

(4) In 'Abstract', the final sentence 'Comparison of CBC- ALYREF to other CBC and ALYREF containing cellular complexes provides insights into the coordinated events during mRNA transcription, splicing, and export' doesn't read smoothly, I would suggest revising it to 'Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insight into the coordinated events during mRNA transcription, splicing, and export.'

We thank the reviewer for the recommendation and have revised accordingly.

(5) In paragraph 'CBC-ALYREF and viral hijacking of host mRNA export pathway', line 6, the sentences preceding and following the term 'However' indicate a progressive or parallel relationship, rather than a transitional one. To enhance the coherence, I would suggest replacing 'However' with 'Furthermore' or 'In addition'.

Corrected in the revised manuscript.

(6) In both Figure 5 and Figure 6, the depicted models are proposed and constructed exclusively through the comparison of the CBC-partial ALYREF with other cellular complexes containing components of CBC and/or ALYREF, which need to be confirmed by more studies. To prevent potential confusion and misunderstandings, it is recommended to replace the term 'model' with 'proposed model'.

Corrected in the revised manuscript.

Reviewer #3 (Recommendations for the Authors):

Major points:

(1) In the Results and Discussion section, the authors mentioned "Recombinant human ALYREF protein was shown to interact with the CBC in RNase-treated nuclear extracts." However, they used mouse ALYREF for cryo-EM investigations. Can the authors include an explanation for this choice during the revision?

In our work, we used a mixture of glutamic acid and arginine to increase the solubility of GSTALYREF. For cryo-EM studies, we use untagged ALYREF to avoid potential issues that may arise from the expression tag. However, untagged ALYREF is less soluble than GST-tagged ALYREF and is not suitable for structural studies in standard buffers. We have made further clarification on this point in this revised manuscript.

(2) In the paragraph on "CBC-ALYREF interfaces", the authors stated "For example, E97 forms salt bridges with K330 and K381 of NCBP1. Y135 on the α2 helix of mALYREF2 makes a hydrogen bond with K330 of NCBP1. The importance of this interface between ALYREF and NCBP1 is highlighted by a K330N mutation found in human uterine corpus endometrial carcinoma." I fail to see a strong connection between their structural observations and previous findings regarding the role of a K330N mutation found in human uterine corpus endometrial carcinoma. The authors should add more words to thread these two parts.

In response to the reviewer’s comment, we now move the discussion of these CBC mutants to the newly added “Conclusion and perspectives” section.

(3) The authors should include side chains of the residues in their figure of Local resolution estimation and FSC curves, especially when they are presenting the binding interface between two components.

We have now included density maps that are overlayed with structural models showing side chains of critical residues. These maps include the NCBP1-mALYREF2 interfaces (Figure 3-figure supplement 1A and 1B), NCBP2-mALYREF2 interface (Figure 3-figure supplement 1C), NCBP1NCBP2 interface (Figure 2-figure supplement 3B and 5B), and the m7G cap region (Figure 2figure supplement 3C and 5C).

Minor points:

(1) Some grammatical mistakes need to be corrected. For example, it is "an mRNA" instead of "a mRNA".

Corrected in the revised manuscript.

(2) The authors can provide more information for the audience to know better about ALYREF when it first appears in the 5th line in the Abstract section. For example, "It promotes mRNA export through direct interaction with ALYREF, a key mRNA export factor, ...".

We have revised the sentence based on the reviewer’s comment.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) RCSB Protein Data Bank. 8SUY
    2. Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. RCSB Protein Data Bank. 8SRR [DOI] [PubMed]
    3. Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. Electron Microscopy Data Bank. EMD-40739 [DOI] [PubMed]
    4. Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) Electron Microscopy Data Bank. EMD-40780

    Supplementary Materials

    Figure 1—source data 1. Original file for the gels in Figure 1B.
    elife-91432-fig1-data1.zip (23.2MB, zip)
    Figure 4—source data 1. Original file for the gels in Figure 4A.
    elife-91432-fig4-data1.zip (24.4MB, zip)
    Figure 4—source data 2. Original file for the gels in Figure 4B.
    elife-91432-fig4-data2.zip (25.4MB, zip)
    MDAR checklist
    elife-91432-mdarchecklist1.docx (87.8KB, docx)

    Data Availability Statement

    The coordinates of CBC-ALYREF and CBC have been deposited in PDB under accession codes 8SRR and 8SUY. The corresponding density maps have been deposited in EMDB under accession codes EMD-40739 and EMD-40780. Source data files have been provided for Figures 1 and 4.

    The following datasets were generated:

    Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) RCSB Protein Data Bank. 8SUY

    Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. RCSB Protein Data Bank. 8SRR

    Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the CBC-ALYREF complex. Electron Microscopy Data Bank. EMD-40739

    Xie Y, Clarke BP, Ren Y. 2024. Cryo-EM structure of the human cap binding complex (CBC) Electron Microscopy Data Bank. EMD-40780

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