An internal ribosome entry site in the coding region of tyrosyl-DNA phosphodiesterase 2 drives alternative translation start

: Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a multifunctional protein that has been implicated in a myriad of cellular pathways. Although most well-known for its phosphodiesterase activity removing stalled topoisomerase 2 from DNA, TDP2 has also been shown to interact with both survival and apoptotic mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, it facilitates enterovirus replication and has been genetically linked to neurological disorders ranging from Parkinson's disease to dyslexia. To accurately evaluate TDP2 as a therapeutic target, we need to understand how TDP2 performs such a wide diversity of functions. Here, we use cancer cell lines modified with CRISPR/Cas9 or stably-expressed TDP2-targeted shRNA and transfection of various TDP2 mutants to show that its expression is regulated at the translational level via an internal ribosome entry site (IRES) that initiates translation at codon 54, the second in-frame methionine of the TDP2 coding sequence. We observed that this IRES drives expression of a shorter, N-terminally truncated isoform of TDP2, ΔN-TDP2, which omits a nuclear localization sequence. Additionally, we noted that ΔN-TDP2 retains phosphodiesterase activity and is protective against etoposide-induced cell death, but co-immunoprecipitates with fewer high-molecular-weight ubiquitinated peptide species, suggesting partial loss-of-function of TDP2's ubiquitin-association domain. In summary, our findings suggest the existence of an IRES in the 5' coding sequence of TDP2 that translationally regulates expression of an N-terminally truncated, cytoplasmic isoform of TDP2. These results shed light on the regulation of this multifunctional protein and may inform the design of therapies targeting TDP2 and associated pathways.