Jana Schmitzová

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.

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.

Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 patients ^1,2 . Molnupiravir increases the frequency of viral RNA mutations ^3,4 and impairs SARS-CoV-2 replication in animal models ^4-6 and in patients ² . Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase (RdRp) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp readily uses the active form of molnupiravir, β-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp progression. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs for transcription.