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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

Schlafen11 (SLFN11) is a relatively unknown human interferon stimulated gene (ISG) whose activity and regulation are poorly understood. SLFN11 is part of the SLFN family of genes, which are primarily found in mammals [1]. The first biological function of SLFN11 was discovered by our lab in 2012, which found that SLFN11 down-regulates the speed at which human immunodeficiency virus (HIV) synthesizes its protein by exploiting HIV’s codon usage bias [2].

In the same year, two separate reports found that SLFN11 sensitizes cancer cells upon exposure to DNA damage agents (DDAs) [3][4], however the mechanism for this sensitization had not been uncovered. In chapter 1 of this dissertation, we show that SLFN11 down-regulates translation of an important DNA damage signaling protein, ataxia telangiectasia and Rad3 related (ATR), as well as prove that the down-regulation of ATR is responsible for the sensitization. We then found that upon treatment of camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor, SLFN11 down-regulates type II transfer RNA (tRNA) (Leucines and Serines) via cleavage. Lastly, SLFN11 appears to inhibit translation of proteins whose codon profile prefer Leu(TTA) and Leu(CTT) codons due to the low abundance of their corresponding tRNA. Our discovery of this novel mechanism is important not only for advancing knowledge of SLFN11’s role in cancer, but also in helping to pave a path in the development of novel chemotherapeutics.

One aspect of SLFN11 that has not been explored and is poorly understood is its regulation. My work seeks to uncover this uncertainty, specifically furthering our understanding of how phosphorylation regulates SLFN11. In chapter 2 of this dissertation, we show three putative phosphorylation sites of SLFN11 via mass spectrometry and mutagenic analysis. At these three sites, SLFN11 loses the ability to down-regulate type II tRNA and subsequently protein translation inhibition when the site is mutated to Aspartic acid (phosphor-mimic), but retains its activity when mutated to Alanine (un-phosphorylatable residue). We further found that the SLFN11 Alanine mutants exploit the codon profile of a protein with complete bias towards Leu(TTA) and Leu(CTT) codons, but not Leu(CTG). This work furthers our knowledge of SLFN11 regulation, and aids in the discovery of the phosphatase(s) and kinase(s) activating or inhibiting SLFN11 respectively.

    Cover page: The Regulation and Activity of Schlafen11
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2019)
    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.
      Cover page: Dephosphorylation activates the interferon-stimulated Schlafen family member 11 in the DNA damage response
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      Transcriptome analysis reveals a strong positive correlation between human Schlafen family member 11 (SLFN11) expression and the sensitivity of tumor cells to DNA-damaging agents (DDAs). Here, we show that SLFN11 preferentially inhibits translation of the serine/threonine kinases ATR and ATM upon DDA treatment based on distinct codon usage without disrupting early DNA damage response signaling. Type II transfer RNAs (tRNAs), which include all serine and leucine tRNAs, are cleaved in a SLFN11-dependent manner in response to DDAs. Messenger RNAs encoded by genes with high TTA (Leu) codon usage, such as ATR, display utmost susceptibility to translational suppression by SLFN11. Specific attenuation of tRNA-Leu-TAA sufficed to ablate ATR protein expression and restore the DDA sensitivity of SLFN11-deficient cells. Our study uncovered a novel mechanism of codon-specific translational inhibition via SLFN11-dependent tRNA cleavage in the DNA damage response and supports the notion that SLFN11-deficient tumor cells can be resensitized to DDAs by targeting ATR or tRNA-Leu-TAA.
        Cover page: DNA damage-induced cell death relies on SLFN11-dependent cleavage of distinct type II tRNAs.
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