Binding of MYC to nascent RNA suppresses innate immune signaling by R-loop-derived RNA-DNA hybrids [seCLIP]

In response to perturbed transcription elongation, the MYC oncoprotein multimerizes and undergoes a phase transition. The underlying mechanisms and their function are unknown. Here, we demonstrate that MYC globally relocalizes from its canonical positions on DNA to nascent RNA in response to the accumulation of intronic RNA. Binding of MYC to RNA induces MYC to form multimers that concentrate the nuclear exosome - a 3'-5' RNA exonuclease - and its targeting complexes around double-stranded RNA and R-loops. MYC harbors four RNA-binding regions (RBRI–IV), which overlap with evolutionarily conserved regions. Of these, RBRIII promotes MYC multimerization and is necessary for recruiting the exosome to R-loops. While RBRIII is dispensable for MYC-dependent transcriptional activation and pancreatic tumor cell proliferation in culture, it is indispensable for sustaining tumor growth in vivo. Via RBRIII, MYC suppresses the accumulation of R-loop-derived RNA-DNA hybrids and prevents them from binding to the pattern recognition receptor TLR3 and activating the innate immune kinase TBK1. Our data demonstrate that MYC has mechanistically distinct functions in transcription and RNA metabolism, and that its phase transition is an RNA-driven stress response that suppresses the accumulation of immunogenic RNA-DNA hybrids. Overall design: A primary objective of the study was to determine whether MYC binds to RNA in cells. To this end, seCLIP experiments were performed in K562 cells (with endogenous MYC) and in U2OSMYC-Tet-On cells that were treated with doxycycline to induce oncogenic MYC levels or with ethanol (EtOH) as a control to represent endogenous MYC levels. To evaluate whether MYC binds to nascent RNA, MYC eCLIPs were performed after CDK9 inhibition and subcellular fractionation into nucleoplasmic and chromatin-associated fractions. Additionally, we sought to determine the effect of chemical treatment with CBL0137, PlaB, or siRNA-mediated EXOSC5 knockout on MYC RNA binding. Furthermore, seCLIP experiments were performed for TLR3 to compare TLR3 RNA binding in KPC cells with and without MYC (shMYC). To demonstrate that RBRIIImut affects MYC RNA binding in cells, we performed MYC eCLIPs in KPC cells that were depleted of endogenous MYC (shMYC) and that had exogenously inducible WTMYC and RBRIIIMUTMYC alleles. A complementary eCLIP aimed to investigate whether MYC RNA binding changes in stressed (CBL0137-treated) U2OS cells. Thus, MYC-AID cells that express doxycycline-inducible, HA-tagged WTMYC and RBRIIIMUTMYC alleles were treated with auxin and doxycycline.