The Tox gene encodes two proteins with distinct and shared roles in gene regulation

Here we report that the murine Tox gene encodes two proteins from a single mRNA and investigate the mechanism of production and function of these proteoforms. The annotated TOX coding sequence is predicted to produce a 526 amino acid protein (TOXFL). However, western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXDN), while the slower migrating band is TOXFL. The TOXDN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL: TOXDN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+8+ cells and subsequent differentiation to CD4+8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXDN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion, than did TOXDN, including uniquely regulated cell cycle and other genes. Naïve CD3+CD8+ splenocytes were isolated from OTI mice and stimulated with OVA peptide for 3 days and transduced to express TOXFL or TOXdeltaN (and IRES-GFP) at 24 hours after initiation of culture. Cells were FACS sorted on day 5 and GFP- (exogenous TOX-) and the top 20% of GFP+ (exogenous TOX+) populations were analyzed by RNA-seq.