Mouse Slfn8 and Slfn9 genes complement human cells lacking SLFN11 during the replication stress response

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Bustos, O. et al. Evolution of the Schlafen genes, a gene family associated with

embryonic lethality, meiotic drive, immune processes and orthopoxvirus

virulence. Gene 447, 1–11 (2009).

Liu, F., Zhou, P., Wang, Q., Zhang, M. & Li, D. The Schlafen family: complex

roles in different cell types and virus replication. Cell Biol. Int. 42, 2–8 (2018).

Schwarz, D. A., Katayama, C. D. & Hedrick, S. M. Schlafen, a new family of

growth regulatory genes that affect thymocyte development. Immunity 9,

657–668 (1998).

Yang, J.-Y. et al. Structure of Schlafen13 reveals a new class of tRNA/rRNAtargeting RNase engaged in translational control. Nat. Commun. 9, 1165 (2018).

Metzner, F. J., Huber, E., Hopfner, K. P. & Lammens, K. Structural and

biochemical characterization of human Schlafen 5. Nucleic Acids Res. 50,

1147–1161 (2022).

Li, M. et al. Codon-usage-based inhibition of HIV protein synthesis by human

schlafen 11. Nature 491, 125–128 (2012).

Ding, J. et al. Schlafen 5 suppresses human immunodeficiency virus type 1

transcription by commandeering cellular epigenetic machinery. Nucleic Acids

Res. 50, 6137–6153 (2022).

Metzner, F. J. et al. Mechanistic understanding of human SLFN11. Nat.

Commun. 13, 5464 (2022).

Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive

modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).

Zoppoli, G. et al. Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes

cancer cells to DNA-damaging agents. Proc. Natl Acad. Sci. USA 109,

15030–15035 (2012).

Gardner, E. E. et al. Chemosensitive relapse in small cell lung cancer proceeds

through an EZH2-SLFN11 axis. Cancer Cell 31, 286–299 (2017).

Zhang, B. et al. A wake-up call for cancer DNA damage: the role of Schlafen

11 (SLFN11) across multiple cancers. Br. J. Cancer 125, 1333–1340 (2021).

Sousa, F. G. et al. Alterations of DNA repair genes in the NCI-60 cell lines and their

predictive value for anticancer drug activity. DNA Repair 28, 107–115 (2015).

Jo, U. et al. SLFN11 promotes CDT1 degradation by CUL4 in response to

replicative DNA damage, while its absence leads to synthetic lethality with

ATR/CHK1 inhibitors. Proc. Natl Acad. Sci. USA 118, e2015654118 (2021).

Mu, Y. et al. SLFN11 inhibits checkpoint maintenance and homologous

recombination repair. EMBO Rep. 17, 94–109 (2016).

Murai, J. et al. SLFN11 blocks stressed replication forks independently of ATR.

Mol. Cell 69, 371–384.e6 (2018).

Murai, J. et al. Chromatin remodeling and immediate early gene activation by

SLFN11 in response to replication stress. Cell Rep. 30, 4137–4151.e6 (2020).

Murai, Y. et al. SLFN11 inactivation induces proteotoxic stress and sensitizes

cancer cells to ubiquitin activating enzyme inhibitor TAK-243. Cancer Res. 81,

3067–3078 (2021).

Okamoto, Y. et al. SLFN11 promotes stalled fork degradation that underlies

the phenotype in Fanconi anemia cells. Blood 137, 336–348 (2021).

Li, M. et al. DNA damage-Induced cell death relies on SLFN11-dependent

cleavage of distinct type II tRNAs. Nat. Struct. Mol. Biol. 25, 1047–1058 (2018).

Lilue, J. et al. Sixteen diverse laboratory mouse reference genomes define

strain-specific haplotypes and novel functional loci. Nat. Genet. 50, 1–16

(2018).

Puck, A. et al. Expression and regulation of Schlafen (SLFN) family members

in primary human monocytes, monocyte-derived dendritic cells and T cells.

Results Immunol. 5, 23–32 (2015).

Neumann, B., Zhao, L., Murphy, K. & Gonda, T. J. Subcellular localization of

the Schlafen protein family. Biochem. Bioph. Res. Co. 370, 62–66 (2008).

Seita, J. et al. Gene Expression Commons: An Open Platform for Absolute

Gene Expression Profiling. Plos One 7, e40321 (2012).

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold.

Nature 596, 583–589 (2021).

ARTICLE

26. Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding

the structural coverage of protein-sequence space with high-accuracy models.

Nucleic Acids Res. 50, D439–D444 (2022).

27. Huang, J. et al. SLFN5-mediated chromatin dynamics sculpt higher-order

DNA repair topology. Mol. Cell 83, 1043–1060 (2023).

28. Yue, T. et al. SLFN2 protection of tRNAs from stress-induced cleavage is

essential for T cell–mediated immunity. Science 372, 6543 (2021).

29. Abdelfattah, N. S. & Mullally, A. Using CRISPR/Cas9 Gene Editing to

Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine

Dependent Hematopoietic Cells. J. Vis. Exp. 131, e56726 (2018).

30. Chaudhuri, A. R. et al. Replication fork stability confers chemoresistance in

BRCA-deficient cells. Nature 535, 382–387 (2016).

31. Quinet, A., Carvajal-Maldonado, D., Lemacon, D. & Vindigni, A. DNA Fiber

Analysis: Mind the Gap! Methods Enzymol. 591, 55–82 (2017).

32. Cong, K. & Cantor, S. B. Exploiting replication gaps for cancer therapy. Mol.

Cell 82, 2363–2369 (2022).

33. Liu, S. et al. RNA polymerase III is required for the repair of DNA doublestrand breaks by homologous recombination. Cell 184, 1314–1329 (2021).

34. Chakraborty, P., Huang, J. T. J. & Hiom, K. DHX9 helicase promotes R-loop

formation in cells with impaired RNA splicing. Nat. Commun. 9, 1–14 (2018).

35. Matsui, M. et al. USP42 enhances homologous recombination repair by

promoting R-loop resolution with a DNA-RNA helicase DHX9. Oncogenesis

9, 1–13 (2020).

36. Nakagawa, K. et al. Schlafen-8 is essential for lymphatic endothelial cell

activation in experimental autoimmune encephalomyelitis. Int. Immunol. 30,

69–78 (2018).

37. Inano, S. et al. RFWD3-mediated ubiquitination promotes timely removal of

both RPA and RAD51 from DNA damage sites to facilitate homologous

recombination. Mol. Cell 66, 622–634 (2017).

38. Qi, F., et al. The ribonuclease domain function is dispensable for SLFN11 to

mediate cell fate decision during replication stress response. Genes Cell, 1–11.

https://doi.org/10.1111/gtc.13056 (2023).

39. Marahatta, A. & Ware, R. E. Hydroxyurea: analytical techniques and

quantitative analysis. Blood Cells, Mol. Dis. 67, 135–142 (2017).

40. Rajkumar, P. et al. Cisplatin concentrations in long and short duration

infusion: implications for the optimal time of radiation delivery. J. Clin. Diagn.

Res. 10, XC01–XC04 (2016).

Acknowledgements

We would like to thank Drs. Junko Murai, Yasuhisa Murai, and Kiichiro Tsuchiya, for

discussions; Dr. Andres Canela for critical reading of the manuscript and discussion; Dr.

Feng Zhang for pX330; Dr. Bruce Beutler for mSLFN2 plasmid; Dr. Hitoshi Kurumizaka

for anti-RAD51 serum; the late Dr. Hiroyuki Miyoshi and RIKEN BRC for the Lentivirus

system; Dr. Koichi Sato for advice for Alfafold 2 structural prediction; Ms. Masami

Tanaka, Mayu Yamabe, Sumiko Matsui, Xuye Wang, and Lin Liu for technical and

secretarial assistance. Anfeng Mu is supported by the Kyoto University Research

Coordination Alliance. This work is also partly supported by the KAKENHI Kiban B

(Grant# 20H03450 to M.T.), Takeda Science Foundation (to A.M.), The Uehara Memorial Foundation (to A.M.), and JSPS Core-to-Core Program (Grant#

JPJSCCA20200009).

Author contributions

A.M., Y.O. and M.T. designed the study. E.A. compared the protein sequences, cloned

the cDNAs and carried out DNA fiber analysis with a help from M.O. and F.Q. A.L.M.

made Ba/F3 Slfn8/9/10 knockout cell lines. Y.K. performed laser track experiments. E.A.,

M.T. and A.M. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s42003-023-05406-9.

Correspondence and requests for materials should be addressed to Anfeng Mu.

Peer review information : Communications Biology thanks the anonymous reviewers for

their contribution to the peer review of this work. Primary Handling Editors: Valeria

Naim and George Inglis. A peer review file is available.

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