Structure of human cytoplasmic Pol II complex explains global transcription repression by GDOWN1.

RNA polymerase II (Pol II) is a 12-subunit enzyme crucial for gene transcription in the nucleus. However, its assembly in the cytoplasm, nuclear import, and nuclear function of assembly factors remain poorly understood. Here, we isolated Pol II from the cytoplasmic fraction of human cells (cfPol II) and find it associated with the assembly and transport factors GDOWN1, RPAP2, GPN1, and GPN3. Cryo-EM analysis of cfPol II resolves RPAP2 and GDOWN1 bound to Pol II at 2.9 Å resolution and shows that Pol II is fully assembled in the cytoplasm before nuclear import. Our structure of GDOWN1 bound to the Pol II surface reveals three distinct regions of GDOWN1 that interact with the RPB2 protrusion domain, RPB3, and RPB10. Biochemical analyses show that GDOWN1 facilitates soluble expression of a subcomplex comprising RPB3, RPB10, RPB11 and RPB12, suggesting a role for GDOWN1 in Pol II assembly. Further, GDOWN1 binding to Pol II overlaps with binding sites of the essential transcription factors IIB and IIF, rendering cfPol II inactive in promoter-dependent transcription initiation in vitro. Our results provide a basis for GDOWN1-dependent global transcription repression and suggest a model for a role of GDOWN1 in Pol II assembly, import, and transcription regulation.