蓬勃发展的表观转录组学

迄今为止,研究者们在RNA上已经发现了百余种不同种类的化学修饰,这些修饰大都分布在丰度较高的非编码RNA中,并对非编码RNA功能的维持具有重要作用.近年来,得益于高分辨率质谱的应用以及全转录组测序技术的开发,越来越多的mRNA上的修饰被发现、精确定量和定位,包括N6-甲基腺嘌呤(m6A)、N6,2-O-二甲基腺嘌呤(m...     

A systematic, ligation-based approach to study RNA modifications

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.     

Epigenetic gene regulation by noncoding RNAs

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.     

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m¹A), N6-methyladenosine (m⁶A), pseudouridine, and 5-methylcytidine (m⁵C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m⁵C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m¹A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m⁶A antibody confirmed the demethylation of m⁶A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m⁶A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.