Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors

Abstract

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation. Protein-protein crosslinking of NTC, functional assays in vitro, and assessment of its role in DNA damage response provide mechanistic insight into the organization of the NTC core and the communication between PLRG1 and Prp19 that enables E3 activity. This reveals a unique mode of regulation for a complex E3 ligase and advances understanding of its dynamics in various cellular pathways.

Ubiquitylation at the Fork: Making and Breaking Chains to Complete DNA Replication

“…Subsequently, RPA ubiquitylation was also shown to be enhanced by camptothecin (CPT; a topoisomerase I inhibitor that creates DSBs at ongoing forks), hydroxyurea (HU), the DNA polymerase inhibitor aphidicolin and the cross-linking agent mitomycin C but not by γ-irradiation (IR) indicating that this modification is particularly relevant to the RSR [ 21 , 67 , 68 ]. Two E3 ubiquitin ligases function on RPA-ssDNA and control RPA ubiquitylation during replication stress: PRP19 and RFWD3 [ 21 , 59 , 67 , 68 , 69 ].…”

“…PRP19 is a multifunctional and essential U-BOX family E3 ubiquitin ligase best known for its role as an evolutionarily conserved pre-mRNA processing factor that functions within the PRP19/CDC5L core complex, an important spliceosome co-factor composed of PRP19, CDC5L, PLRG1 and BCAS2 [ 70 , 71 , 72 , 73 , 74 ]. In addition to its U-BOX E3 ligase domain, PRP19 also contains a central coiled-coil domain which allows its tetramerization and a C-terminal substrate recognition WD40-repeat module [ 69 , 75 , 76 ]. It was first discovered in yeast as a mutant that accumulates intron-containing pre-mRNA at non-permissive temperature and was later shown to physically associate with the spliceosome and promote its activation [ 77 , 78 , 79 ].…”

“…Knockdown (KD) of the core subunits of the PRP19/CDC5L complex also results in sensitivity to the replication stress-inducing agents mitomycin C, UV and HU [ 84 , 85 ]. Support for a more direct link between the PRP19 complex and the RSR came from the discovery that the complete PRP19/CDC5L core complex relocates onto RPA-ssDNA upon replication stress or at resected DSBs [ 21 , 69 , 86 ]. On RPA-ssDNA, the PRP19 complex promotes ATR activation.…”

“…Indeed KD of PRP19, CDC5L, PLRG1 or BCAS2 all strongly decrease RSR signaling by ATR and the recruitment of this master checkpoint kinase to stalled forks as measured by phosphorylation of its substrates (RPA and/or CHK1) and by ATRIP foci formation [ 21 , 69 , 84 , 86 ]. Depletion of PRP19 and other splicing factors was also shown to impede DSB resection which may contribute to ATR-activation and RPA phosphorylation defects [ 21 , 69 , 87 , 88 ]. Furthermore, PRP19 complex KD impedes replication fork restart and HR indicative of extensive roles in the RSR [ 21 , 87 , 89 ].…”

“…Mechanistically, PRP19 KD decreases RPA70 and RPA32 ubiquitylation upon CPT treatment and this can be complemented by the re-expression of WT PRP19 but not by ubiquitin ligase or RPA-binding mutants [ 21 , 59 ]. More recent data indicates that RPA ubiquitylation also depends on PLRG1 which is required to activate the E3 ligase activity of the PRP19/CDC5L core complex [ 69 ]. Thus, in response to replication stress, the PRP19 complex transforms into a sensor of RPA-ssDNA and functions as a ubiquitin ligase on RPA-ssDNA to promote RSR signaling and replication fork repair.…”