Identification and characterization of small NAD-capped RNAs in yeast

RNA是生物学中一个重要的调节因子。它的简单结构通过各种化学修饰得到了增强。最近，我们发现普遍存在的氧化还原辅酶烟酰胺腺嘌呤二核苷酸(NAD)以帽状方式与细菌中一组特定的调控RNA结合，并调节这些RNA的功能。此外，我们还发现了第一个原核解离酶--nudC。在之前的资助期间，我们开发了用于定量分析NAD-RNA的新的质谱学和电泳法，以及它们产生的制备方法。我们检测并定量了酵母、拟南芥和小鼠中的NAD-RNA。将NAD CaptureSeq应用于四个不同的酵母总RNA样品，我们发现大部分mRNAs都是富集的。另一个大的富集组是小RNA。酵母中NAD的解离是由NudC同系物Npy1催化的，它在标准的m7G帽上是不活跃的。NAD-RNA在原核生物和真核生物中的存在表明了一种更普遍的现象，这种现象可能在所有生命王国中都是保守的。本项目的目的是对酵母中的NAD-caped-RNA进行鉴定。在这里，我们建议在模式生物酿酒酵母中识别、量化和表征小的5‘-NAD封顶的RNA。为了发现这样的RNA，我们将改进NAD CaptureSeq，以专门识别小于25个核苷酸的NAD上限RNA。对于选定的修饰RNA，我们将揭示其生物学意义和生物合成。我们将讨论NAD封端的RNA的类型和序列，修饰对环境条件的依赖，生物发生以及体外和体内的解帽。对于这些研究，我们可以使用我们小组已经建立的一系列方法。

RNA is an important regulator in biology. Its simple architecture is augmented by diverse chemical modifications. Recently we discovered the ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) to be attached to a specific set of regulatory RNAs in bacteria in a cap-like manner, and to modulate the functions of these RNAs. Furthermore, we discovered the first prokaryotic decapping enzyme, NudC. In the previous funding period we developed new mass-spectrometric and electrophoretic methods for the quantitative analysis of NAD-RNA, as well as preparative methods for their generation. We detected and quantified NAD-RNA in yeast, Arabidopsis, and mouse. NAD captureSeq was applied to four different yeast total RNA samples, and we found that mostly mRNAs are enriched. Another large enriched group are small RNAs. NAD decapping in yeast is catalysed by the NudC homolog Npy1, which is inactive on the canonical m7G cap. The existence of NAD-RNA in prokaryotic as well as eukaryotic organisms points to a more general phenomenon that might be conserved in all kingdoms of life. The aim of this project is to proceed with the identification of NAD-capped-RNA in yeast. Here, we propose to identify, quantify, and characterize small 5’-NAD capped RNAs in the model organism Saccharomyces cerevisiae. To discover such RNAs we will refine the NAD captureSeq to specifically identify NAD-capped RNAs that are smaller than 25 nucleotides. For selected modified RNAs we will unravel the biological significance and biosynthesis. We will address the types and sequences of NAD-capped RNAs, the dependence of the modification on environmental conditions, the biogenesis as well as the decapping in vitro and in vivo. For these studies, we can make use of a range of methods already established in our group.