Yang Jun, Sharma Sunny, Kötter Peter, Entian Karl-Dieter
Institute of Molecular and Cellular Microbiology, Goethe University, Frankfurt am Main, Germany 60438.
Institute of Molecular and Cellular Microbiology, Goethe University, Frankfurt am Main, Germany 60438
Nucleic Acids Res. 2015 Feb 27;43(4):2342-52. doi: 10.1093/nar/gkv058. Epub 2015 Feb 4.
Methylation of ribose sugars at the 2'-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2'-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5' central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D' box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.
核糖在2'-羟基处的甲基化是核糖体RNA（rRNA）中主要的化学修饰之一，由小核仁RNA（snoRNA）指导的C/D盒小核仁核糖核蛋白颗粒（snoRNPs）催化。此前对C/D盒snoRNAs的生化和计算分析已经鉴定并定位了rRNAs中大量的2'-羟基核糖甲基化。在本研究中，我们使用绿豆核酸酶保护试验和反相高效液相色谱（RP-HPLC）系统地分析了酿酒酵母中18S rRNA的核糖甲基化。出乎意料的是，我们在酵母18S rRNA 5'中央结构域的螺旋18中的G562位置鉴定出一种迄今未知的核糖甲基化。此外，我们鉴定出snR40负责引导snoRNP复合物催化G562核糖甲基化，这使其成为迄今为止已知的第二种靶向三个核糖甲基化位点的snoRNA：18S rRNA中的Gm562、Gm1271以及25S rRNA中的Um898。我们对snR40的序列和突变分析表明，snR40对Gm562和Gm1271甲基化使用相同的D'盒和甲基化指导序列。随着Gm562及其相应snoRNA的鉴定，18S rRNA的核糖甲基化及其相应snoRNAs的完整集合最终得以确立，为理解这些修饰的生理功能开辟了广阔前景。
