Structural basis of catalytic activation in human splicing

Abstract

Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated Bact and the branching-competent B* spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16)1–3. However, because PRP2 is observed only at the periphery of spliceosomes3–5, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic6,7. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate—the BAQR complex—found halfway through the catalytic activation process. The cryogenic electron microscopy structure of BAQR reveals how PRP2 and Aquarius remodel Bact and BAQR, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.

Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins

“…DIM1, together with PRP8, are displaced from the 5′ss GU dinucleotide during the transformation of the B complex into the B act complex, and the 5′ss is handed over to the RNF113A and SF3A2 proteins (reviewed in Kastner et al, 2019 ). The latter, as well as proteins that sequester the pre-mRNA branch site adenosine (the nucleophile for the first catalytic step of splicing), are then displaced during B* formation by the action of the helicase PRP2 (Bai et al, 2020 ; Schmitzova et al, 2023 ), enabling the first step of pre-mRNA splicing. Thus, prior to step 1, the 5′ss is sequestered by different proteins, preventing the former from premature nucleophilic attack.…”

Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3′ splice site usage

“…In addition to DDX41/SACY-1, metazoans have four more helicases involved in splicing that are not conserved in S. cerevisiae ( De Bortoli et al 2021 ). In Schizosaccharomyces pombe , helicases Prp2 and Aquarius act sequentially to activate the spliceosome, whereas in S. cerevisiae , Prp2 performs activation without an Aquarius homolog ( Schmitzová et al 2023 ). The spliceosome may have divided helicase duties among an increasing number of helicases as it evolved, and PRP22 and DDX41 may likewise share a role in activating the second step of splicing.…”

The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

“…Our findings in interaction and quantitative TMT-proteomics analyses revealing that USP7 interacts with and regulates the abundance of small nuclear ribonucleoproteins, the RNA helicase protein Dhx, and the RNA regulatory protein Fam172a 38,39 , further support a role for USP7 in the control of RNA processing. Recent studies suggest that Ppil4 is recruited to active spliceosomes to facilitate catalytic activation of the spliceosome by positioning the key RNA helicase Prp2 downstream of the branching site adenosine of introns 40,41 . It will be important to determine how USP7 regulation of Ppil4 ubiquitination might influence the catalysis of RNA splicing in neurons.…”