Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21 (RNA-Seq). Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21 (RNA-Seq)

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternate pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduce DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi. Overall design: We performed Total RNA-seq for RIP-seq input. polyA-enriched, polyA-depleted RNA-seq to check the expression of snoRNA and their host genes and LucKD, DDX21 KD polyA enriched RNAseq to check host gene expression upon DDX21 KD.

PARP抑制剂（PARP inhibitors, PARPi）通过诱导DNA修复缺陷（例如BRCA1/2突变细胞）相关的合成致死效应，阻断癌细胞增殖。我们在BRCA1/2基因完整的乳腺癌细胞中，发现了一条PARPi介导的生长调控替代通路，该通路涉及核糖体DNA（ribosomal DNA, rDNA）转录与核糖体生物发生。
PARP-1可结合小核仁RNA（small nucleolar RNAs, snoRNAs），后者在不依赖DNA损伤的情况下激活核仁内的PARP-1催化活性。活化的PARP-1对RNA解旋酶DDX21进行ADP核糖基化（ADP-ribosylation）修饰；DDX21定位于核仁，当其被ADP核糖基化修饰后可促进rDNA转录。
采用PARPi处理或对ADP核糖基化位点进行突变，会降低DDX21的核仁定位效率、rDNA转录水平、核糖体生物发生进程、蛋白质翻译速率以及细胞增殖能力。该通路的核心特征在小鼠异种移植瘤模型与人类乳腺癌患者样本中均得到验证。PARP-1与核仁定位的DDX21高表达与癌症相关不良预后显著相关。本研究为DNA修复无缺陷但生长受PARPi抑制的癌细胞中，PARPi的抗肿瘤疗效提供了机制层面的理论依据。

实验设计：我们对RNA免疫沉淀测序（RNA Immunoprecipitation sequencing, RIP-seq）的输入样本开展总RNA测序；通过聚腺苷酸化富集RNA测序、聚腺苷酸化去除RNA测序，检测snoRNA及其宿主基因的表达水平；同时对荧光素酶敲降（LucKD）组与DDX21敲降组样本进行聚腺苷酸化富集RNA测序，以探究DDX21敲降后宿主基因的表达变化。