CDK12 and CDK13 suppress distinct intronic polyadenylation sites

Abstract

Cyclin-dependent kinases CDK12 and CDK13 are RNA polymerase II C-terminal domain Ser2 kinases that regulate transcriptional elongation and co-transcriptional RNA processing. Loss-of-function (LOF) mutations in these kinases are observed in multiple cancers and are characterized by increased intronic polyadenylation (IPA), which generates prematurely terminated transcripts and can give rise to novel intronic peptides. To investigate the distinct contributions of CDK12 and CDK13 to suppressing early transcriptional termination, we modeled selective kinase inhibition in OVCAR4 ovarian cancer cells using THZ531, a dual CDK12/13 inhibitor. A CRISPR-engineered CDK12 C1039S mutation conferred THZ531 resistance, enabling functional distinction between CDK12 and CDK13 LOF conditions. Using Poly(A) Click-seq (PACseq) across a six-point inhibitor dose curve, we identified thousands of IPA sites with distinct dose-response behaviors, allowing us to classify CDK12- and CDK13-dependent subsets. Oxford Nanopore long-read sequencing confirmed premature termination events and revealed full-length isoform switches, demonstrating how IPA alters coding potential by truncating protein domains and producing novel, intronically encoded peptide sequences. In parallel, integration of long-read transcriptomes with mass spectrometry data identified peptides derived from IPA isoforms, confirming that intronic sequences can be translated. Together, these findings establish distinct roles for CDK12 and CDK13 in maintaining transcript integrity, demonstrate that IPA transcripts are not only abundant but also translationally competent, and provide evidence for a mechanistic link between transcriptional dysregulation and the generation of novel peptide isoforms in CDK12/13-deficient cancers. These results highlight a potential therapeutic vulnerability through the exploitation of IPA-derived peptides as tumor-specific neoantigens.