Sequential verification of transcription by Integrator and Restrictor [ZC3H4_mNET-seq]

The decision between productive elongation and premature termination of promoter-proximal RNA polymerase II (RNAPII) is fundamental to metazoan gene regulation. Integrator and Restrictor complexes are implicated in promoter-proximal termination, but why metazoans utilise two complexes and how they are coordinated is unknown. Here, we show that Integrator and Restrictor sequentially and non-redundantly monitor distinct early steps of transcription. Integrator predominantly engages paused RNAPII to trigger premature termination, which can be prevented by cyclin-dependent kinase 7/9 activity. Crucially, we reveal a previously unrecognised and universal checkpoint imposed by Restrictor after promoter-proximal pausing. This restriction zone presents an additional opportunity to verify RNAPII. Progression through this second checkpoint requires U1 small nuclear ribonucleoprotein (snRNP), which antagonises Integrator and Restrictor in a U1-70K dependent manner. These findings reveal the principles of a sequential validation mechanism governing the balance between productive and attenuated transcription, rationalising the necessity of Integrator and Restrictor complexes in metazoans. Overall design: Expression profiling by mammalian Native Elongating Transcript sequencing (mNET-seq) for ZC3H4-DHFR degron cell line (with or without TMP)