The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

Abstract

Prp19/Pso4, a U-box containing E3 ligase, has a demonstrated role in pre-mRNA splicing, but has also been implicated in both yeast and mammalian cells as having a direct role in DNA damage processing. In this report we provide further evidence in support of this latter assertion. We show that hPrp19 forms an ubiquitylated oligomeric species that is resistant to disruption by SDS gel electrophoresis under nonreducing conditions suggesting that is mediated by a thiolester between ubiquitin and a cysteine residue in Prp19. The level of this species is significantly enhanced upon treatment of cells with DNA damaging agents, and its association with chromatin is increased. In addition, hPrp19 is known to form a stable core complex with Cdc5L, Plrg1, and Spf27; however, ubiquitylated hPrp19 fails to interact with either Cdc5L or Plrg1 indicating that DNA damage can induce profound alterations to the hPrp19 core complex. Finally, we show that overexpression of hPrp19 in human cells provides a pro-survival affect in that it reduces the levels of apoptosis observed after exposure of cells to DNA damage.The pso4-1 mutant of S. cerevisiae was isolated in a genetic screen for strains sensitive to the cross-linking agent psoralen plus UVA (PUVA) [1,2]. Characterization of this mutant indicated that it was particularly sensitive to bifunctional alkylating agents, but was also sensitive to a broad range of DNA damaging agents including IR, UV, and monofuntional alkylating compounds. Induced mutagenesis and induced and spontaneous mitotic recombination were all greatly reduced in this mutant suggesting that Pso4 is involved in a recombinational pathway of error-prone repair. Based on epistasis analysis PSO4 was assigned to both the yeast RAD6 and RAD52 groups indicating the pleiotropic nature of this mutation [3,4]. Interestingly, cloning of PSO4 showed that it was allelic to PRP19 a previously characterized component of the pre-mRNA splicing complex in both yeast and human cells [5][6][7][8][9][10].The complete function of Prp19 in mRNA splicing is not known, however, it has been shown that the Prp19p-associated complex is required for activation of the pre-mRNA splicesome and for the stable association of the small nuclear RNAs U5 and U6 with the spliceosome after U4 is dissociated [11,12]. These findings suggest a possible structural role for the Prp19-associated complex in pre-mRNA splicing. Mammalian Prp19 associates with a large number of splicing factors, although, it appears to form a core complex with three other proteins including Cdc5L, Plrg1, and Spf27 [9]. The structure of the Prp19 core complex is not completely resolved, however, it has been shown that yeast Prp19 forms a tetramer via its coiled coil domains, and that the tetramer interacts with one copy of Cdc5L through the associated coiled coil domains *Corresponding author. Fax. 713-792-1474; E-Mail: rlegersk@mdanderson.org. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65–Prp19 complex

“…The Prp19 antibody detected additional bands (most prominently in S-100) (Fig. 2B, lanes 1,2) that may correspond to previously described ubiquitinated forms of Prp19 (Lu and Legerski 2007). Consistent with that report, which showed that Prp19 ubiquitination disrupts its association with other complex members, these modified forms failed to cofractionate with other PRP19C subunits on Mono Q (data not shown), suggesting that the modification disrupts interactions between Prp19 and other complex subunits.…”

Death by UVC Light Correlates with Protein Damage in Isogenic Human Tumor Cells: Primary Tumor SW480 versus its Metastasis SW620

“…Possible reasons for the increased sensitivity of the particular cell line should first be sought among the functions of those proteins. Interestingly, TGM2 and PRP19 have previously been shown to promote cancer cell survival when overexpressed [39,40], which is consistent with our results. Namely, high level of carbonylation has been shown to inactivate protein function, thus, rendering the cells deprived of the particular protein activity (similarly to a conditional mutant).…”

Human Pso4 Is a Metnase (SETMAR)-binding Partner That Regulates Metnase Function in DNA Repair

“…HPso4 is a human homolog of the PS04/PRP19 gene in S. cerevisiae that has pleiotropic functions in DNA recombination and errorprone repair (15,16,18,28,(33)(34)(35)(36)(37)(38). Specific role(s) for Pso4 in DSB repair is not known; however, it has been identified as a component of the nuclear matrix (20).…”

“…Because the Pso4 is a part of the pre-mRNA splicing complex consisting of Pso4, Cdc5L, Plrg1, and Spf27 (17), Metnase may interact with a portion of the pre-mRNA splicing complex. In fact, the presplicing complex was shown to link to DNA repair through a direct physical interaction of Cdc5L with WRN, a DSB repair factor deficient in Werner syndrome (18,28). It is possible that the MetnasehPso4 interaction is only a part of the much bigger complex that participates in DSB repair in vivo.…”

“…Based on a proteomic analysis, the 55-kDa protein was identified as the human homolog of the PRP19 gene product of S. cerevisiae (PRP19/Pso4, NCBI accession number NP_055317) involved in pre-mRNA splicing and DNA repair (16,18,28), and the 50-kDa protein was identified as NP_006506, a cleavage product of Metnase (SETMAR). Interestingly, the 37-kDa was identified as the human homolog of Spf27 (NP_060544), a member of the Prp19 core complex involved in pre-mRNA splicing (18,28). Because both Metnase and hPso4 are involved in DSB repair (1, 16), we examined whether Metnase directly interacts with hPso4.…”