Elongation factor-specific capture of RNA polymerase II complexes

Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation factor-specific mNET capture (EMCAP) of RNA polymerase II complexes for sequencing and mass spectrometry analysis, for investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply EMCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3'end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3'end processing factors but depleted of splicing components. Importantly, the EMCAP-seq profiles of SCAF4- and SCAF8-RNAPII complexes reflect their function as mRNA-anti-terminators and their competing functions at the end of genes where they prevent or promote transcriptional readthrough. Overall design: Comparison of mammalian native transcript elongation (mNET) capture of RNA polymerase II complexes associated with SCAF4 and SCAF8 in HEK293 cells.