Fig. 6: KSHV PAN RNA assists in the recruitment of ATAT1 to NAT10. | Cell Death & Differentiation

Fig. 6: KSHV PAN RNA assists in the recruitment of ATAT1 to NAT10.

From: Lactylation of NAT10 promotes N4‐acetylcytidine modification on tRNASer-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

Fig. 6

A The iSLK-KSHV cells transduced with lentiviral NAT10 (NAT10-Myc) or control (pCDH) for 48 h were treated with or without doxycycline (Doxy) for 72 h, and then subjected to anti-Myc IP assay and analyzed by Western blot with the anti-ATAT1 antibody. B, C The iSLK-KSHV cells with doxycycline induction for 72 h were subjected to the anti-ATAT1 (B), anti-NAT10 (C), or immunoglobulin G (Anti-IgG) RNA immunoprecipitation, and the precipitated PAN RNA were examined by RT-qPCR. ***, P < 0.001 by Student’s t-test versus the IgG group. D HEK293T cells with NAT10 overexpression (NAT10-Flag) were transduced with lentiviral PAN (pHAGE-PAN) or control (pHAGE) for 48 h. Cells were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-ATAT1 and anti-Pan Kla antibodies. E The iSLK-KSHV cells with NAT10 overexpression (NAT10-Myc) were transduced with lentiviral PAN shRNAs (shPAN-1, shPAN-2, shPAN-3, and shPAN-4) or control (shCtrl) for 48 h. Cells were subjected to the anti-Myc immunoprecipitation and analyzed by Western blot using anti-ATAT1 and anti-Pan Kla antibodies. F The iSLK-KSHV cells transduced with lentiviral NAT10 (NAT10-Myc) or control (pCDH) for 48 h were treated with or without doxycycline (Doxy) for 72 h, while the induced NAT10-overexpressing cells were further treated with RNase. Cell proteins were subjected to anti-Myc IP assay and detected by Western blot with anti-ATAT1 and anti-Pan Kla antibodies. G Schematic illustration for the mechanism of lactylated NAT10-dependent tRNASer-CGA-1-1 ac4C modification during KSHV reactivation. As for NAT10 lactylation, ATAT1 serves as a writer. During KSHV reactivation, PAN RNA assists in the interaction between ATAT1 and NAT10 to promote the lactylation level of NAT10. Lactylated NAT10 occurring on Lys290 mediates tRNASer-CGA-1-1 ac4C modification, and eventually increases the translation efficiency of a series viral lytic transcripts, resulting in progeny virion production and virus reactivation.

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