Cytoplasmic NAD+ Synthesis and Ribosomal Protein Mono(ADP-Ribosyl)ation Maintain Proteostasis in Ovarian Cancer

Global protein synthesis is upregulated in cancers, and thus cancer cells are prone to errors in translation, which may lead to protein misfolding and proteotoxic stress. The pathways that maintain protein homeostasis support the transformation and growth of cancer cells. Here, we show that the cytosolic NAD+ synthesis pathway (controlled by NMNAT-2) plays an essential role in ovarian cancer by maintaining protein homeostasis by regulating the activity of PARP-16, that MARylates the ribosomal proteins. Using polysome-RNA Seq analysis, we found that inhibiting ribosomal protein MARylation by knocking down NMNAT-2 or PARP-16 leads to increased association of specific mRNAs with polysomes and enhanced translation of a set of mRNAs that contain specific secondary structures in their 3’UTRs, which leads to protein aggregation. Collectively, our study demonstrates that NMNAT-2 regulates PARP-16 activity and ribosomal protein MARylation, which control protein homeostasis by fine-tuning the levels of protein synthesis in ovarian cancers. To evaluate the changes in mRNA loading on polysomes when NMNAT-2 or PARP-16 are knockdown, we performed polysome-RNA-Seq analysis on these cells. We isolated total RNA (Input RNA) and polysome-associated RNA from OVCAR3 cells subjected to NMNAT2 or PARP16 knockdown and performed RNA-Seq analysis. Please note that each processed data was generated from both replicates and is linked to the corresponding replicate1 sample records.

癌症中整体蛋白质合成过程上调，因此癌细胞更易出现翻译错误，进而引发蛋白质错误折叠与蛋白毒性应激。
维持蛋白质稳态（protein homeostasis）的通路可支持癌细胞的转化与生长。
本研究发现，由NMNAT-2调控的胞质NAD+合成通路，通过调控PARP-16的活性维持蛋白质稳态，从而在卵巢癌中发挥关键作用——PARP-16可对核糖体蛋白进行单ADP核糖基化（MARylation）修饰。
本研究利用多聚核糖体RNA测序（polysome-RNA Seq）分析发现，通过敲低NMNAT-2或PARP-16抑制核糖体蛋白的单ADP核糖基化修饰后，特定mRNA与多聚核糖体的结合增多，且一类在3'非翻译区（3’UTRs）携带特定二级结构的mRNA的翻译效率显著提升，最终引发蛋白质聚集。
综上，本研究证实，NMNAT-2通过调控PARP-16活性与核糖体蛋白的单ADP核糖基化修饰，精准调控卵巢癌中的蛋白质合成水平，进而维持蛋白质稳态。
为评估敲低NMNAT-2或PARP-16后，多聚核糖体结合的mRNA负载变化，本研究对上述细胞开展了多聚核糖体RNA测序分析。
本研究从经NMNAT2或PARP16敲低处理的OVCAR3细胞中分离了总RNA（Input RNA）与多聚核糖体结合RNA，并开展了RNA测序（RNA-Seq）分析。
请注意，所有已处理数据均来自两份生物学重复，并与对应的重复1样本记录相关联。