Abstract 13: A disease-linked mutant reveals a critical N-terminal influence of SOX2 on translation

Abstract Changes in cell fate are primarily attributed to the spatio-temporal influence of transcription factors, but the ultimate level of expression control remains translation. Some transcription factors (such as YB-1, RfaH and Bicoid) play complementary roles in translation, and we have recently reported that also the pluripotency-inducing transcription factor SOX2 has ribosomal affinity and modulates transcript selection (Schaefer et al., Cell Reports 2024). Although this post-transcriptional layer of regulation mainly reflects SOX2‘s C-terminal influence in the cytosol, we here present evidence for a further involvement also of N-terminal SOX2 in translation.Ribosomal affinity within N-terminal parts of SOX2 first showed in domain separation analyses in breast cancer-derived cell lines. However, although SOX2 fragments (1-119) and (1-179) co-sedimented with 60S ribosomes in sucrose density gradients, they imposed no translational fingerprint in polysome profiles as otherwise seen for full-length or C-terminal SOX2. Nonetheless, the synthesis of bulk protein (indicated by OPP incorporation into nascent polypeptides) did change and hundreds of mRNAs experienced a translational regulation upon SOX2 N-terminus induction. Among these, 65 differentially regulated factors stood out as co-regulated by full-length or N-terminal SOX2, but unaltered or counter-regulated in SOX2 C-terminus induced cells. Overrepresentation analyses within STRING and TISSUES databases indicated a preferential expression in the reproductive tract for 35 of these hits, and also under more stringent inclusion criteria, a functional assignment to the reactomes mitosis and reproduction was reconfirmed. In phenotypic correlation, we indeed noted a co-regulation of cell growth in full-length or N-terminus induced T47D and MDA-MB468 reporter cells, while the C-terminus recapitulated the proliferation rate of control cells. Real-life evidence for a SOX2 N-terminal contribution to translation, however, came from a disease-linked human mutant, SOX2(L97P), originally identified in the context of eye development. When expressed in reporter cells the L97P mutation severely impaired 60S binding, so that neither the polysome profile nor the phosphorylation status of RPS6 changed as otherwise seen for wild-type SOX2.Our study highlights that, despite decades of preceding research, the protein biochemistry of even key pluripotency factors remains underexplored and still holds unforeseen learning. In summary, we present here extended evidence for a translational significance of SOX2 that (i) involves N-terminal contributions, (ii) arises in physical contact with ribosomes, and (iii) takes influence on cell proliferation. A better understanding of the underlying molecular principles may thus pave the way to a targeted control of cell division, selectively in SOX2-driven reprogramming and cancer. Citation Format: Meric Ataman, Nitish Mittal, Hui Wang, Sergiy Velychko, Tata Nageswara Rao, Jordan Löliger, Jonas Lötscher, Sarah Roffeis, Christoph Hess, Hans R. Schöler, Ed Hurt, Matthias P. Wymann, Mihaela Zavolan, Claudia Lengerke, Thorsten Schaefer. A disease-linked mutant reveals a critical N-terminal influence of SOX2 on translation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 13.