Molecular basis of polyadenylated RNA fate determination in the nucleus

Main

RNA polymerase II (RNAPII) extensively transcribes mammalian genomes, yielding a wide range of unadenylated and polyadenylated RNAs 1 , 2 , 3 , 4 . Moreover, individual transcription units that generate standard full-length transcripts also give rise to an array of shorter isoforms 9 , 10 . Thus, functional RNAs are produced alongside a wealth of futile RNAPII products. Whereas mature functional pA + RNAs, such as protein-coding mRNAs, are exported from the nucleus to the cytoplasm, their non-functional counterparts are typically retained and degraded 3 , 4 . This is primarily achieved by the nucleoplasmic PAXT connection, which consists of a heterodimeric core of the RNA helicase MTR4 and the Zn-finger protein ZFC3H1 8 , 11 . Additional, and less well-described, interactions with the nuclear pA + RNA-binding protein PABPN1 and other transiently interacting RNP components, sometimes referred to as extended PAXT components, may aid in directing transcript turnover by the 3′−5′ exonucleolytic exosome complex 12 , 13 , 14 , 15 . However, how these interactions provide a biochemical basis by which PAXT distinguishes non-functional pA + RNAs remains a major unresolved question.

Prior to their nuclear export, pA + RNAs are packaged with proteins into pA + RNPs. Central to this process is the export factor and DExD-box ATPase UAP56, which is recruited to pA + RNPs in preparation for their nuclear export 5 , 16 . At the nuclear envelope, the activity of the nuclear pore complex (NPC)-associated GANP–PCID2–SEM1 (GANP–PS) trimer of TREX-2 6 facilitates the release of RNA from UAP56, enabling export 7 , 17 . Again, how pA + RNP sorting is orchestrated to favour the selected export of functional pA + RNAs is unknown.

Here we interrogate two TREX-2-like human complexes, SAC3D1–PCID2–SEM1 (SAC3D1–PS) and LENG8–PCID2–SEM1 (LENG8–PS), in which the conserved SAC3D1 and LENG8 proteins, respectively, replace GANP. The GANP–PS, SAC3D1–PS and LENG8–PS complexes are structurally similar and share the ability to release UAP56 from RNA. Notably, we show that LENG8–PS offers PAXT a module, that acts on UAP56 to promote transcript turnover in contrast to the RNA export activity of TREX-2. Our findings reveal that nuclear pA + RNA export and decay utilize a shared biochemical mechanism to act on pA + RNPs but with fundamentally different outcomes. Based on the substrate preference of PAXT and its separate nuclear localization from TREX-2, we propose a general model for pA + RNP fate determination.

TREX-2-like complexes release RNA from UAP56

To mediate the docking of export-competent pA + RNPs at the NPC, UAP56 binds the five subunit TREX-2 complex 7 (GANP, PCID2, SEM1, CETN2 or CETN3, and ENY2). Within this complex, UAP56 contacts the TREX-2 complex core (TREX-2 M ), which comprises PCID2, SEM1 and the SAC3 domain of the scaffolding subunit GANP 7 (Fig. 1a , left). The ability of TREX-2 M to release UAP56 from the pA + RNP depends on the conserved ‘wedge loop’ within the SAC3 domain 7 , 17 (Fig. 1b ). Notably, similar SAC3 domains are found in the UAP56-interacting LENG8 and SAC3D1 proteins 7 . Although they are broadly conserved amongst eukaryotes, these proteins share no sequence features with GANP or each other aside from the SAC3 domain (Fig. 1a,b ). Moreover, proteome-wide AlphaFold2 screens suggested that SAC3 domains of LENG8 or SAC3D1 form complexes with PCID2 and SEM1 18 , 19 , 20 , thus mimicking TREX-2 M . Finally, and central to the present study, LENG8 co-immunoprecipitated with PAXT core components ZFC3H1 and MTR4 8 , 21 (also see Fig. 2 below) and was shown to interact with PCID2 and SEM1 in both human and yeast cells 22 , 23 . Collectively, this prompted us to investigate these TREX-2 M -like complexes in more detail.

Fig. 1: TREX-2-like complexes bind UAP56 to trigger RNA release. The alternative text for this image may have been generated using AI.

Full size image

a , Cartoons of TREX-2 and TREX-2-like core complexes (top) and their domain architectures (bottom). PCID2, dark blue; SEM1, mid blue; SAC3 domain-containing proteins GANP, LENG8 and SAC3D1, shades of blue. Wedge loop positions are shown as grey bars. Regions included in the atomic models in e , f are indicated by black lines. WH, winged helix. b , Multiple sequence alignment of wedge loop sequences of human GANP (UniProt O60318), LENG8 (UniProt Q96PV6) and SAC3D1 (UniProt A6NKF1), with a conserved tyrosine residue anchoring the wedge loop on the SAC3 domain (Y anchor ) and the central wedge loop arginine (R wedge ) highlighted. Colouring by conservation (blue letters, conserved residue; blue background, invariant residue). c , d , UAP56 RNA release assay, demonstrating the stimulatory effects of LENG8–PS M ( c ) or SAC3D1–PS M ( d ) complexes. Bead-immobilized 15 poly-uridine RNA was incubated with UAP56 and ATP to form UAP56–ADP-P i –RNA complexes 7 and subsequently challenged with recombinant LENG8–PS M or SAC3D1–PS M complexes, or their respect…