Structural Basis of 7SK RNA 5′-Gamma-Phosphate Methylation and Retention by MePCE

Abstract

7SK non-coding RNA is an essential regulator of eukaryotic transcription elongation through dynamic RNP assemblies that sequester or release the positive transcription elongation factor b (P-TEFb). All 7SK RNPs include a core comprising 7SK RNA, methylphosphate capping enzyme (MePCE), and La-related protein 7 (Larp7). In addition to its non-enzymatic role as a core component of 7SK RNPs, MePCE catalyzes the formation of a gamma-phosphate monomethylation cap, unique to 7SK and U6 in humans. How MePCE can specifically recognize and modify its RNA substrate and remain bound to 7SK to form the core 7SK RNP has not been previously known. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. We find that 7SK recognition is achieved by an extensive network of protein contacts to a 5' hairpin-single-stranded RNA region, involving the triphosphate group, two terminal basepairs and six single-stranded nucleotides, thus explaining MePCE specificity for 7SK and U6. The large size of the RNA binding site required for recognition and catalysis is unique among RNA methyltransferases studied to date. The structures reveal SAH and product RNA in a near-transition state geometry, where the cofactor SAH is buried inside the active site by the RNA-interacting residues. Unexpectedly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. Taken together, these data describe the features of an unusual RNA capping enzyme, and reveal the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of the core 7SK RNP.