Sam68 stimulates Theta-Mediated End-Joining by suppressing poison exon inclusion in Polq mRNA

DNA polymerase Theta-Mediated End-Joining (TMEJ) is a stand-alone mutagenic DNA double-strand break (DSB) repair pathway that becomes critical when high-fidelity repair is compromised. Although the enzymatic mechanism of TMEJ has been extensively studied in recent years, its regulation remains poorly understood. Here, we identify and characterize the RNA-binding protein Sam68 (also known as Khdrbs1) as a key modulator of TMEJ in mammalian cells. We demonstrate that Sam68 is required for proper expression of Polq, the gene encoding polymerase theta (Polθ), the central factor in TMEJ. Loss of Sam68 results in reduced TMEJ at CRISPR-induced DSBs and increased sensitivity to ionizing radiation. Mechanistically, Sam68 promotes correct splicing of Polq mRNA by suppressing the inclusion of a conserved poison exon that introduces a premature termination codon in the transcript. This function depends on the RNA-binding domain of Sam68. Genetic deletion of the poison exon restores Polq expression and rescues TMEJ activity in Sam68-depleted cells. Together, these findings establish alternative splicing as a direct mechanism controlling TMEJ capacity and reveal a conserved regulatory mechanism that tunes mutagenic DSB repair through modulation of Polθ abundance.