Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The total RNAs from iSLK-KSHV NAT10 +/- cells (NAT10 +/- ) and iSLK-KSHV NAT10 +/+ cells (WT) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. B The heatmap for differentially expressed tRNAs in cells shown as in ( A ) by tRNA acRIP-seq analysis. The pseudo-color represented as the fold enrichment (IP/Input) of WT and NAT10 +/− group, respectively. Any tRNAs not detected in tRNA acRIP-seq were plotted as white in the heatmap. C The total RNAs from cells shown as in ( A ) were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Ser-CGA-4-1 , tRNA Leu-TAG-3-1 , tRNA Ser-AGA , tRNA Leu-TAA , and U6 as the control. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( A ). *** P < 0.001 by Student’s t -test. E Schematic representation of the acetylation site mutation of tRNA Ser-CGA-1-1 (Upper) and tRNA Leu-TAG-3-1 (Lower). The C (marked in red) at position 12 was mutated to U (marked in dark blue). F The total RNAs from iSLK-KSHV cells with overexpression of the wild type tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (WT), mutant tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (Mut), and their control empty vector ( EV ) for 48 h were respectively subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Leu-TAG-3-1 and U6 as the control. G . The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with overexpression of the wild type (tRNA Ser-CGA-1-1 -WT) or its mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h, and their control (tRNA-EV). *** P < 0.001 by Student’s t -test. H The expression levels of vIRF1 and NAT10 in iSLK-KSHV cells with overexpression of the wild type (WT), mutant (Mut) tRNA Ser-CGA-1-1 , and their control (EV) for 48 h were detected by Western blot. I The SDS gel analysis of the in vitro translation reaction supplemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 , in comparison with reaction mixtures that not containing mRNA (No mRNA). J By the assessment of ORF26, real-time DNA-PCR for cells shown as in ( H ) was performed to detect viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Dot Blot, Staining, Control, Northern Blot, Quantitative RT-PCR, Mutagenesis, Over Expression, Plasmid Preparation, Expressing, Western Blot, SDS-Gel, In Vitro, Comparison

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A Schematic representation of DUF1726 domain (marked in blue), RNA helicase domain (marked in yellow), N -acetyltransferase domain (marked in green) and RNA-binding domain (marked in purple) of NAT10. The identified lactylation lysine site ( K ) within the RNA helicase domain was marked in red, which was mutated to arginine ( R ) and marked in dark blue. B The iSLK-KSHV cells transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h were subjected to the anti-Flag immunoprecipitation, and then examined by Western blot with the anti-Pan Kla antibody. C The sequences around NAT10 Lys290 from different species were aligned. Conserved Lys290 was marked in red. D The total RNAs from cells shown as in ( B ) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. E The iSLK-KSHV cells with THUMPD1 overexpression (THUMPD1-Myc) were transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h. Cells were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-Myc antibody. F The total RNAs from iSLK-KSHV cells transduced with the wild type (NAT10) and the mutant (K290R) NAT10 for 48 h were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , and U6 as the control. G The expression levels of vIRF1 and K-bZIP in cells treated as in ( B ) were examined by Western blot with the corresponding antibodies. H The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( B ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. I The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with the wild type (NAT10-WT) or the mutant (NAT10-K290R) NAT10 overexpression, which were complemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h. * P < 0.05, **, P < 0.01 and *** P < 0.001 by Student’s t -test. J By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( B ) was performed to examine viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: RNA Binding Assay, Transduction, Mutagenesis, Control, Immunoprecipitation, Western Blot, Dot Blot, Staining, Over Expression, Northern Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The expression levels of vIRF1 and K-bZIP in iSLK-KSHV cells transduced with lentiviral sgATAT1 (sgATAT1 #2, sgATAT1 #3) or control lentivirus (Lenti-V2) for 72 h were examined by Western blot with the corresponding antibodies. B The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in cells treated as in ( A ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. C By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( A ) was performed to examine viral copy number after doxycycline stimulation for 72 h. ***, P < 0.001 by Student’s t -test. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in iSLK-KSHV cells transduced with lentiviral ATAT1 (ATAT1) or its control (pCDH) for 48 h. ***, P < 0.001 by Student’s t -test. E By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( D ) was performed to detect viral copy number after doxycycline stimulation for 72 h. ** P < 0.01 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Expressing, Transduction, Control, Western Blot, Quantitative RT-PCR