1 research outputs found
Lysines in the RNA Polymerase II C‑Terminal Domain Contribute to TAF15 Fibril Recruitment
Many cancer-causing chromosomal translocations
result in transactivating
protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity
(LC) domains fused to a DNA binding domain from one of several transcription
factors. Recent work demonstrates that higher-order assemblies of
FET LC domains bind the carboxy-terminal domain of the large subunit
of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins
may mediate aberrant transcriptional activation by recruiting RNA
polymerase II to promoters of target genes. Here we use nuclear magnetic
resonance (NMR) spectroscopy and hydrogel fluorescence microscopy
localization and fluorescence recovery after photobleaching to visualize
atomic details of a model of this process, interactions of RNA pol
II CTD with high-molecular weight TAF15 LC assemblies. We report NMR
resonance assignments of the intact degenerate repeat half of human
RNA pol II CTD alone and verify its predominant intrinsic disorder
by molecular simulation. By measuring NMR spin relaxation and dark-state
exchange saturation transfer, we characterize the interaction of RNA
pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2
LC domains and observe that heptads far from the acidic C-terminal
tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of
CTD lysines in heptad position 7 to consensus serines reduced the
overall level of TAF15 fibril binding, suggesting that electrostatic
interactions contribute to complex formation. Conversely, mutations
of position 7 asparagine residues and truncation of the acidic tail
had little effect. Thus, weak, multivalent interactions between TAF15
fibrils and heptads throughout RNA pol II CTD collectively mediate
complex formation