NADcapPro Seq and CircNAD –Methods for Accurate Profiling of NAD and Non-Canonical RNA Caps in Eukaryotes

Accurate identification of NAD-capped RNAs is essential for understanding their biological function. Previous transcriptome-wide methods used to profile NAD-capped RNAs contain inherent limitations of having hindered the accurate identification of NAD caps from eukaryotic RNAs. Herein we introduced two novel orthogonal methods to precisely identify NAD-capped RNAs. One is D-SPAAC, a copper-free click-chemistry-based approach, and the second is an intramolecular ligation-based circNAD to resolve implicit limitations of the previous methods, which enabled us to unravel unforeseen features of NAD RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs are different, and 3) NAD caps can be added post-transcriptionally. Moreover, we uncovered a dichotomy of NAD RNAs in translation where NAD RNAs are detected with mitochondrial ribosomes but not cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.准确识别NAD加帽RNA（NAD-capped RNAs）对于解析其生物学功能至关重要。此前用于表征NAD加帽RNA的全转录组方法存在固有局限，妨碍了从真核生物RNA中精准鉴定NAD加帽结构。本文中，我们开发了两种全新的正交方法以精准识别NAD加帽RNA：其一为基于无铜点击化学的D-SPAAC方法，其二为基于分子内连接的circNAD分析策略，以解决既往方法的隐性局限，借此揭示酿酒酵母中NAD RNA的未被预见的特征。与既往研究报道相悖的是，我们发现：1）细胞内的NAD RNA可为全长聚腺苷酸化转录本；2）NAD加帽RNA与经典m7G加帽RNA的转录起始位点存在差异；3）NAD加帽结构可通过转录后方式添加。此外，我们还揭示了NAD RNA在翻译过程中的二分特性：NAD RNA可与线粒体核糖体结合，但无法与细胞质核糖体结合，这表明其倾向于在线粒体中完成翻译。