Abstract
Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus's distinct codon preference. Additionally, lack of SLFN11 expression has been linked to the resistance of cancer cells to DNA-damaging agents (DDAs). We recently resolved the underlying mechanism, finding that it involves SLFN11-mediated cleavage of select tRNAs predominantly employed in the translation of the ATR and ATM Ser/Thr kinases, thereby establishing SLFN11 as a novel tRNA endonuclease. Even though SLFN11 is thus involved in two of the most prominent diseases of our time, cancer and HIV infection, its regulation remained thus far unresolved. Using MS and bioinformatics-based approaches combined with site-directed mutagenesis, we show here that SLFN11 is phosphorylated at three different sites, which requires dephosphorylation for SLFN11 to become fully functionally active. Furthermore, we identified protein phosphatase 1 catalytic subunit γ (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. In summary, our work has identified both the mechanism of SLFN11 activation and PPP1CC as the enzyme responsible for its activation. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivates SLFN11.
Highlights
Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus’s distinct codon preference
Monitoring levels of the HIV p24 capsid protein derived from the gag gene revealed that changing Ser-210 or Thr-220 to either Ala or Asp yielded SLFN11 mutants that failed to suppress p24 protein synthesis, hinting that these amino acid changes likely interfere with structural elements of SLFN11
It is noteworthy that the SLFN11 mutants that failed to limit p24 synthesis were expressed at substantially higher levels than functional SLFN11 proteins (Fig. 1b), the variability among the different SLFN11 mRNA levels is insignificant (Fig. S1a), suggesting that SLFN11 translation itself is to some extent self-regulated
Summary
Human Schlafen 11 (SLFN11) is an interferon-stimulated gene (ISG) that we previously have demonstrated to ablate translation of HIV proteins based on the virus’s distinct codon preference. We identified protein phosphatase 1 catalytic subunit ␥ (PPP1CC) as the upstream enzyme whose activity is required for SLFN11 to cleave tRNAs and thereby act as a selective translational inhibitor. Our findings open up future studies of the PPP1CC subunit(s) involved in SLFN11 activation and the putative kinase(s) that inactivates SLFN11. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. SLFNs share no significant sequence similarity with other proteins besides a weakly conserved NH2-terminal putative ATPase associated with diverse cellular activities (AAA) domain. Slfn1-knockout mice displayed no apparent phenotype, whereas in striking contrast, forced transgenic expression of Slfn[1] in T cells greatly reduced T-cell growth and development, with a reduction in the number of thymocytes to 1–30% compared with normal mice (3)
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