Intragenic recruitment of NF-kB drives splicing modifications upon activation by the oncogene Tax of HTLV-1

Chronic NF-kB activation in inflammation and cancer has long been linked to persistent activation of NF-kB–responsive gene promoters. However, NF-kB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-kB factor RELA to intragenic regions regulates alternative splicing upon NF-kB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-kB activation–dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-kB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-kB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-kB–related diseases. Overall design: HEK 293T-LTR-GFP cells (Delebecque et al., 2002) containing an integrated GFP reporter gene under the control of the TAX-responsive HTLV-1 LTR were transiently transfected with siRNAs against DDX5 and DDX17 (GGCUAGAUGUGGAAGAUGUdTdT) (or siRNA control siGL2 (CGUACGCGGAAUACUUCGAdTdT)) and/or expression vectors pSG5M-TAX or pSG5M empty. PolyA transcripts were extracted at 48h post-transfection and RNA-seq analyses were performed as previously described (Lambert et al., 2018). RNA-seq libraries were generated at the Aros Applied Biotechnology (Aarhus, Denmark) using Stranded mRNA Sample Prep kit (Illumina) and sequenced using the illumina HiSeq 2500 technology. RNA-seq data were analyzed using FaRLine with the FasterDB database (Benoit-Pilven et al., 2018; Mallinjoud et al., 2014).