Elongation factor-specific capture of RNA polymerase II complexes
Research output: Contribution to journal › Journal article › Research › peer-review
Documents
- Fulltext
Final published version, 3.65 MB, PDF document
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 (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP 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 ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.
Original language | English |
---|---|
Article number | 100368 |
Journal | Cell Reports Methods |
Volume | 2 |
Issue number | 12 |
DOIs | |
Publication status | Published - 2022 |
Bibliographical note
Publisher Copyright:
© 2022 The Authors
- CP: molecular biology, mNET-seq, nascent transcription, RNA polymerase II, RNAPII, RNAPII CTD-associated factors, RNAPII transcript elongation, SCAF4, SCAF8, termination, transcription
Research areas
ID: 330894273