Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.
(Molecular Cell 77, 1322–1339.e1–e11; March 19, 2020) After discussion among the co-authors, who are all in agreement, Amit Kumar’s contribution to this paper warranted inclusion as a co-author. The authors regret this omission. The paper has since been corrected online, including the author list, acknowledgments, and author contributions, which also appear here. We acknowledge Andreas Schlosser at the mass spectrometry technology platform at Rudolf-Virchow-Zentrum, Würzburg, for the FLAG-NUAK1 and the label-free proteomic experiments. We thank Ulrike Samfass for technical support. This work was supported by grants to M.E. from the European Research Council (AuroMYC), the Federal Ministry of Education and Research (DKTK Therapeutic Targeting of MYC), and the German Research Foundation via the Research Group FOR2314 (Therapeutic Windows) and via grant SFB1309 (to Y.-C.C. and B.K.). G.C. performed most experiments. I.R. and G.C. performed PLAs. C.S.-V. prepared samples for SILAC mass spectrometry. G.M., A.K., S.K., and R.S.V. processed and analyzed SILAC mass spectrometry. Y.-C.C. performed and analyzed TMT mass spectrometry. F.P., L.W., and D.M. designed, developed, and tested the specificity of the BAY-880 compound. A.B. and C.P.A. sequenced ChIP-seq, ChIP-RX, and RNA-seq libraries. S.W. and A.B. analyzed ChIP-seq and RNA-seq data. G.C., S.K., E.W., C.G., R.L., B.K., and M.E. devised, supervised, and interpreted experiments. G.C. and M.E. wrote the paper. Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of TranscriptionCossa et al.Molecular CellJanuary 31, 2020In BriefTumors displaying high MYC levels depend on the NUAK1 kinase. Cossa et al. show that NUAK1 binds protein phosphatase 1 (PP1) and its regulatory subunit PNUTS. They show that this complex associates with the splicing machinery and provides a feedback control of transcription that can be overridden by deregulated MYC. Full-Text PDF Open Access
Highlights•N-MYC forms complexes with TFIIIC, RAD21, and TOP2A•TFIIIC recruits RAD21 and is required for N-MYC-dependent pause release of Pol II•Aurora-A displaces TFIIIC, TOP2A, and RAD21 from N-MYC during S phase•Aurora-A inhibits pause release of Pol II during S phaseSummaryMYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.Graphical abstract
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