Sequences of CRISPR guides and RT-PCR primers.

Paralogs often exhibit functional redundancy, allowing them to effectively compensate for each other’s loss. However, this buffering mechanism is frequently disrupted in cancer, exposing unique paralog-specific vulnerabilities. Here, we identify a selective dependency on the splicing factor TRA2A. We find that TRA2A and its paralog TRA2B are synthetic lethal partners that function as widespread and largely redundant activators of both alternative and constitutive splicing. While loss of TRA2A alone is typically neutral due to compensation by TRA2B, we discover that a subset of cancer cell lines are highly TRA2A-dependent. Upon TRA2A depletion, these cell lines exhibit a lack of paralog buffering specifically on shared splicing targets, leading to defects in mitosis and cell death. Notably, TRA2B overexpression rescues both the aberrant splicing and lethality associated with TRA2A loss, indicating that paralog compensation is dosage-sensitive. Together, these findings reveal a complex dosage-dependent relationship between paralogous splicing factors, and highlight how dysfunctional paralog buffering can create a selective dependency in cancer.