Elongation factor-specific capture of RNA polymerase II complexes
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Elongation factor-specific capture of RNA polymerase II complexes. / Gregersen, Lea H.; Mitter, Richard; Svejstrup, Jesper Q.
In: Cell Reports Methods, Vol. 2, No. 12, 100368, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Elongation factor-specific capture of RNA polymerase II complexes
AU - Gregersen, Lea H.
AU - Mitter, Richard
AU - Svejstrup, Jesper Q.
N1 - Publisher Copyright: © 2022 The Authors
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - CP: molecular biology
KW - mNET-seq
KW - nascent transcription
KW - RNA polymerase II
KW - RNAPII
KW - RNAPII CTD-associated factors
KW - RNAPII transcript elongation
KW - SCAF4
KW - SCAF8
KW - termination
KW - transcription
U2 - 10.1016/j.crmeth.2022.100368
DO - 10.1016/j.crmeth.2022.100368
M3 - Journal article
C2 - 36590686
AN - SCOPUS:85144433719
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
SN - 2667-2375
IS - 12
M1 - 100368
ER -
ID: 330894273