Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome
Jens F. Sundström1,9, Alena Vaculova2,9, Andrei P. Smertenko3,9,10, Eugene I. Savenkov1,9, Anna Golovko1,9, Elena Minina1,9, Budhi S. Tiwari1, Salvador Rodriguez-Nieto2, Andrey A. Zamyatnin Jr1, Tuuli Välineva4, Juha Saarikettu4, Mikko J. Frilander5, Maria F. Suarez6, AntonZavialov7, Ulf Ståhl1, Patrick J. Hussey3, Olli Silvennoinen4,8, Eva Sundberg1, Boris Zhivotovsky2 and Peter V. Bozhkov1,10
Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals1,2. Plantslack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases3,4. Although metacaspases are essential for PCD5–7, their natural substrates remain unknown4,8. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads toactivation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression9–15, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our resultsestablish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN. Gene expression is controlled by specialized sets of proteinsfunctioning through several pathways at transcriptional and post-transcriptional levels. One of these proteins, TSN, functions in activating transcription9,10,13,
subsequent mRNA splicing 15, regulation of RNA silencing (as a component of the RNA-induced silencing complex, RISC)11 and in the pathway that edits and destroys double-stranded RNA12,14. The ability of TSN to perform several functions isattributed to its complex domain structure. TSN is composed of a single Tudor domain and five staphylococcal nuclease (SN)-like domains (Fig. 1a; Supplementary Information, Fig. S1)16. Sequence analysis of Homo sapiens TSN (HsTSN) protein revealed a DAVD 790 motif, a known consensus cleavage site for caspase-3-like enzymes17,18, between the Tudor and the fifth SN (SN5) domains (Fig. 1a;Supplementary Information, Fig. S1). Correspondingly, caspase-3 processed recombinant HsTSN into large (relative molecular mass of about 90,000, Mr 90K) and small (about 10K) fragments, and inhibition of caspase-3 with DEVD-CHO abolished the cleavage (Fig. 1b). Aminoterminal sequencing of the fragments confirmed the predicted cleavage of HsTSN after the DAVD 790 motif. Induction of apoptosis in HCT116colon carcinoma cells with 5-fluorouracil (5FU) led to increased caspase-3-like activity and cleavage of endogenous HsTSN into two fragments with the same molecular mass, as observed in vitro (Fig. 1b, c). This effect was not cell- and drug-specific because a similar pattern of endogenous HsTSN cleavage was detected in camptothecin (CPT)-treated HeLa cells (Fig. 1d). As the proteolysis of HsTSN didnot occur either when the P1 position of the DAVD motif was mutated (TSND790E) or after treatment with the pan-caspase inhibitor zVAD-fluoromethylketone (zVAD-fmk; Fig. 1d, e), we conclude that HsTSN is a new component of the human PCD degradome and is cleaved by caspase-3 between the Tudor and SN5 domains. Next, the physiological significance of HsTSN proteolysis was examined using cells...