In metazoans, RNA polymerase II transcribed immature U-rich small nuclear RNAs (snRNAs), ultimately destined to be incorporated into spliceosomal particles, are exported within a specific export complex from the nucleus to the cytoplasm, where further processing of the snRNA occurs. In the nucleus, nascent snRNAs are bound via their 5′ m7G caps to the nuclear cap-binding complex (CBC, a heterodimer of CBP80 and CBP20). A protein called PHAX (‘phosphorylated adapter for export’) then links the cargo, CBC-bound snRNA, to the CRM1 nuclear export receptor, which is bound to its essential cofactor RanGTP. Although the snRNA-CBC-PHAX-CRM1-RanGTP export complex was first characterized 25 years ago (Figure 1a and b) [1], there are several long-standing questions that we sought to address by determining its structure and then using the structure to perform mutagenesis studies with a functional readout. Firstly, how exactly does PHAX, a 394 residue, largely unstructured protein (apart from a small RNAbinding domain) interact with both CBC and CRM1 and potentially the snRNA as well? Secondly, how can one explain the synergistic assembly of the complex, which requires mutual interaction of all components to be stable? Thirdly, why does PHAX have to be specifically phosphorylated (hence its name) by casein kinase II on an N-terminus proximal serine-rich region (known as ST2) for complex stability and export functionality?
We in vitro assembled the human snRNA export complex from its recombinant components (notably in vitro phosphorylated PHAX) and determined the structure at around 2.5 Å resolution using cryo-EM data collected on the Krios CM01 at the ESRF operated by the PSB partners (Figure 1c) [2]. Reconstructions using full length U1 snRNA or with a short capped RNA gave similar results, showing that under these conditions only the cap structure makes interactions within the complex. The structure reveals that a central region of PHAX (residues 112-162) becomes fully ordered. This includes a long helix (residues 112-136), whose N-terminal end binds CBC and C-terminal end, containing the nuclear export sequence (NES), binds CRM1, thus directly bridging the cargo and export receptor moieties of the complex. Interestingly, Trp116 of PHAX binds into the so-called Trp-binding pocket of CBC that is competitively used by other CBC binding effector molecules [3]. In addition, other conserved residues of PHAX directly reinforce the already strong binding by CBC of the 5′ cap, with notably R147 and Y154 sandwiching the second base in the dinucleotide cap structure. Finally, what about PHAX phosphorylation? Supported by confident AlphaFold predictions, we identified from the cryo-EM density, a short peptide of PHAX, containing the conserved 57-YR sequence, bound to a distant region of CRM1. This is immediately upstream of the ST2 phosphorylated region (residues 64-76, with up to four serines phosphorylated) of PHAX, which we predict to make a ‘fuzzy’ electrostatic interaction with the well-characterized, neighboring positively charged surface of CRM1/RanGTP (Figure 2). This surface is implicated in direct binding of other, potentially competing, RNA cargoes in tRNA, microRNA or HIV Rev-RRE RNP export complexes [4].
To test the importance of these interactions on complex stability, collaborators at the University of Aarhus developed a cellular assay involving degron-mediated knockdown of the endogenous PHAX and its replacement by structure-based point mutants. A proximity labeling pulldown was then used to identify the recruitment of proteins to mutated PHAX, compared to wild-type. Whereas the mutation W116A in PHAX abolished assembly of the entire complex in vivo, individual PHAX mutations R147A and Y154E maintained PHAX-CBC binding but prevented recruitment of CRM1-RanGTP. Mutagenesis of PHAX R58E or the ST2 phosphorylation region abolished complex assembly. These structural and mutagenesis results reveal the sensitivity of the snRNA export
complex stability to each individual interaction mediated by PHAX, highlighting its role as a molecular glue and explaining the synergistic nature of functional complex assembly.
S. Cusack (EMBL), J. Kadlec (IBS), E. Dubiez (EMBL and IBS)
[1] Ohno M, Segref A, Bachi A, Wilm M et al. (2000) Cell, 101, 187-198
[2] Dubiez E, Garland W, Finderup Brask M, Boeri Erba E et al. (2025) Nat Struct Mol Biol, 32, 1555-1566
[3] Dubiez E, Pellegrini E, Finderup Brask M, Garland W et al. (2024) Cell Reports, 43, 11639
[4] Smith A, Li Y, Velarde A, Cheng Y et al. (2025) Molecular Cell, 85, 3108-3122
Figure 1: (a) Schematic of snRNA nuclear export [1]. (b) Functional regions of PHAX, (c) Cryo-EM derived structure of the snRNA nuclear export complex.
Figure 2: Interaction of the PHAX ST2 phosphorylated region with the prominent basic surface of CRM1-RanGTP, is anchored by 57-YR of PHAX binding to CRM1.