MYCN is an RNA-binding accessory protein of the nuclear exosome. MYCN is an RNA-binding accessory protein of the nuclear exosome

The MYCN oncoprotein broadly binds promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3’-5’exoribonuclease complex, to facilitate progression through the S phase hinting at an RNA-centric function. Here we show that MYCN forms stable high molecular weight complexes with multiple RNA-binding proteins including the nuclear exosome. RNA-binding assays reveal that MYCN directly binds to thousands of predominantly intronic RNA sites via a short, highly conserved sequence motif termed MYCBoxI. Exosome depletion results in MYCN globally re-localizing from promoters to intronic RNAs. The vacant promoters are then occupied by the MXD transcription repressor protein MNT, which inhibits MYCN-dependent transcription. MYCN fosters the degradation of its bound introns via tight association with the nuclear exosome targeting complex (NEXT). Our data demonstrate that MYCN is an RNA-binding protein that controls RNA turnover and argue that the competition between MYCN’s RNA- and DNA-bound states links the dynamics of the MYCN/MAX/MXD network to mRNA processing. Overall design: CUT&RUN, ChIP and ChIP-Rx DNA-sequencing was done with SH-EP NMYC-ER cells where the NMYC expression can be induced with 4-OHT. For all CUT&Run sequencing and some ChIP-seq and ChIP-Rx sequencing experiments, SH-EP NMYC-ER cells were used that carry a doxycycline inducible shRNA against TF3C5. For CUT&Run sequencing samples, the immunoprecipitation of digested DNA was done for EXOSC5 with or without NMYC expression and with or without expression of shTF3C5. As control, DNA was sequenced following immunoprecipitation with an unspecific IgG antibody. For ChIP-seq and ChIP-Rx sequencing experiments, the immunopresipitated antibodies are listed below. All experiments were done with or without NMYC expression and where indicated, a shRNA targeting TF3C5 was induced via doxycycline or one of the below listed inhibitors was applied.

MYCN癌蛋白与其伴侣蛋白MAX形成异二聚体，广泛结合基因组启动子区域。MYCN还可与核外切体（nuclear exosome，一种3’-5’外核糖核酸酶复合物）相互作用，以促进细胞S期进程，这暗示其存在以RNA为中心的功能。本研究表明，MYCN可与包括核外切体在内的多种RNA结合蛋白形成稳定的高分子量复合物。RNA结合实验显示，MYCN通过一段短且高度保守的序列基序（命名为MYCBoxI），直接结合数千个主要定位于内含子区域的RNA位点。核外切体敲低会导致MYCN从启动子区域整体重新定位至内含子RNA。空出的启动子区域随后会被MXD转录抑制蛋白MNT占据，进而抑制MYCN依赖的转录过程。MYCN通过与核外切体靶向复合物（nuclear exosome targeting complex, NEXT）紧密结合，促进其结合的内含子的降解。我们的数据证实MYCN是一类调控RNA周转的RNA结合蛋白，并指出MYCN的RNA结合与DNA结合状态之间的竞争，可将MYCN/MAX/MXD网络的动态变化与mRNA加工过程相联系。整体实验设计：本研究使用SH-EP NMYC-ER细胞系开展CUT&RUN、ChIP与ChIP-Rx DNA测序实验，该细胞系中NMYC的表达可通过4-OHT诱导。对于所有CUT&RUN测序实验，以及部分ChIP-seq和ChIP-Rx测序实验，我们采用了携带针对TF3C5的多西环素（doxycycline）诱导型短发夹RNA（shRNA）的SH-EP NMYC-ER细胞。针对CUT&RUN测序样本，我们分别在有或无NMYC表达、有或无shTF3C5表达的条件下，对EXOSC5进行消化后DNA的免疫沉淀。作为对照，我们使用非特异性IgG抗体进行免疫沉淀后对DNA进行测序。对于ChIP-seq和ChIP-Rx测序实验，免疫沉淀所用抗体详见下述。所有实验均在有或无NMYC表达的条件下开展；若有标注，可通过多西环素诱导靶向TF3C5的shRNA表达，或加入下述所列的一种抑制剂。