Molecular Basis of Transcription-Coupled Pre-mRNA Capping

Capping is the first step in pre-mRNA processing, and the resulting 5′-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5′-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5′ end. Thus, the RNA 5′ end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5′-exonucleases during early transcription. [Display omitted] •Stable binding of capping enzyme to Pol II requires RNA with a 5′-triphosphate•Architecture of Pol-II-capping enzyme complex is derived from cryo-EM and crosslinking•Capping enzyme binds at RNA exit tunnel, explaining seamless RNA protection Co-transcriptional capping is the first step in pre-mRNA processing and required for mRNA stability and translation. Martinez-Rucobo et al. show that the capping enzyme binds around the RNA exit tunnel of RNA polymerase II to ensure seamless RNA protection.