Sam68 stimulates polymerase theta-mediated end-joining by suppressing poison exon inclusion in Polq mRNA.

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 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. Mechanistically, Sam68 promotes correct splicing of Polq messenger RNA by suppressing the inclusion of a conserved poison exon (PE) that introduces a premature termination codon in the transcript. This function depends on the RNA-binding domain of Sam68. Genetic deletion of the PE 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.