Decapping enzyme NUDT12 partners with BLMH for cytoplasmic surveillance of NAD-capped RNAs

RNA polymerase II transcripts receive a protective 5ʹ m7G cap early during transcription. An alternative cap can be acquired when RNA pol II initiation uses the redox cofactor nicotinamide adenine dinucleotide (NAD) to generate NAD-capped RNAs. The biology of mammalian NAD-RNAs, including its turnover and physiological roles are not completely understood. Here we identify NUDT12 as a cytosolic decapping enzyme for NAD-RNAs. Structural, biochemical and functional studies reveal how homodimerization modulates specificity of NUDT12 for NAD-RNAs as substrate. NUDT12 is localized within a few discrete cytoplasmic granules that are distinct from P-bodies. We identity Bleomycin hydrolase (BLMH) as a factor that associates with NUDT12 in a large ~600 kDa dodecamer complex, and we demonstrate that BLMH is required for correct localization of NUDT12 into these granules. This complex is functional in human cell cultures, as artificial tethering of BLMH to a reporter mRNA results in downregulation of reporter expression, similar to that seen with tethering of NUDT12. Analysis of mouse liver transcriptome from the Nudt12 knockout mouse reveals that a select set of RNAs are regulated, including a significant upregulation of circadian clock transcripts. Given that NAD biosynthesis is under circadian control, our study points to a physiological role for the cytosolic surveillance of NAD-RNAs. RNA sequencing was used to identify transcript abundance differences between Nudt12-/- and Nudt12+/+ in mouse liver and kidney. To characterize the effect of NUDT12 absence on circadian gene expression, the RNA sequencing was used to compare gene expression in relation to dark-light cycle.RNA聚合酶II (RNA polymerase II)转录的产物在转录早期会获得具有保护作用的5'端7-甲基鸟苷帽 (m⁷G cap)。当RNA聚合酶II起始过程利用氧化还原辅因子烟酰胺腺嘌呤二核苷酸 (NAD)时，可生成带有NAD帽的RNA，从而获得另一种帽结构。目前对于哺乳动物NAD帽RNA (NAD-RNAs)的生物学特性，包括其周转代谢与生理功能，尚未完全阐明。本研究鉴定出NUDT12是一种靶向NAD-RNAs的胞质脱帽酶。结构生物学、生物化学与功能实验揭示了同源二聚化如何调控NUDT12对底物NAD-RNAs的识别特异性。NUDT12定位于若干离散的细胞质颗粒中，该结构与P小体 (P-bodies)存在显著差异。我们鉴定出博莱霉素水解酶 (Bleomycin hydrolase, BLMH)是一种与NUDT12结合的蛋白因子，二者可形成分子量约600 kDa的十二聚体复合物；实验证实BLMH对于NUDT12精准定位于上述细胞质颗粒是必需的。该复合物在人类细胞培养体系中具有生物学功能：将BLMH人工锚定至报告基因mRNA可导致报告基因表达下调，这一现象与锚定NUDT12时的效果一致。对Nudt12基因敲除小鼠的肝脏转录组分析显示，有特定子集的RNA的表达受到调控，其中包括生物钟转录本的显著上调。鉴于NAD的生物合成受昼夜节律调控，本研究提示胞质对NAD-RNAs的监控机制具有生理意义。本研究采用RNA测序 (RNA sequencing)技术，鉴定了Nudt12基因敲除 (Nudt12-/-)与野生型 (Nudt12+/+)小鼠肝脏和肾脏中转录本丰度的差异。为了阐明NUDT12缺失对昼夜节律基因表达的影响，我们同样采用RNA测序技术，比较了不同明暗周期下的基因表达谱。