NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap(4)A) and Down-Regulates Immune Response and Cancer Promotion Genes

Marriott, Andrew S.; Vasieva, Olga; Fang, Yongxiang; Copeland, Nikki A.; McLennan, Alexander G.; Jones, Nigel J.

The RNA-Seq analysis was validated by performing real-time qRT-PCR on a selection of genes representing various affected pathways (Fig 3) . These results confirmed the direction of regulation (up or down) for all genes studied. The magnitude of change was also similar for the majority of genes, with a correlation coefficient of 0.83 between the two data sets (Fig 3, inset) . However, for some genes with a zero value of FPKM for one of the samples in the RNA-Seq analysis, the use of the pseudocount method by edgeR to calculate a fold-change has led to a significantly different value, e.g. GFRA1 and TNF. Nevertheless, the values calculated by edgeR are used in the following discussions as they are available for all genes and are still a good relative indication of the change in expression. In order to show that the observed differential gene expression correlates solely with increased Ap 4 A rather than the related ADPR-Ap 4 A derivatives, qRT-PCR analysis was also performed with RNA extracted from NuKO cells grown in the presence of 100 nM KU-0058948, a PARP1 and PARP2 inhibitor that prevents the synthesis of ADPR-Ap 4 A species [17] . The results were very similar to those obtained in the absence of KU-0058948 (Fig 3) , showing that, for these genes at least, ADPR-Ap 4 A is not the cause of the differential expression. The function of ADPR-Ap 4 A, if any, is still unclear.