Jia Dong, Cai Lun, He Housheng, Skogerbø Geir, Li Tiantian, Aftab Muhammad Nauman, Chen Runsheng
Bioinformatics Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
BMC Mol Biol. 2007 Sep 29;8:86. doi: 10.1186/1471-2199-8-86.
The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans.
The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins.
Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs.
2,2,7-三甲基鸟苷（TMG）帽结构是具有关键细胞功能的非编码RNA（ncRNA）的重要功能特征，如一些小核RNA（snRNA）。在这里，我们使用K121和R1131抗TMG抗体进行免疫沉淀，以系统鉴定秀丽隐杆线虫中所有目前已鉴定的ncRNA的TMG帽结构。
两种抗TMG抗体沉淀出了一组相似的秀丽隐杆线虫ncRNA。沉淀物中发现了所有已知具有TMG帽结构的snRNA，这表明我们的鉴定系统是有效的。沉淀物中还发现了与剪接相关的其他ncRNA家族，如SL RNA和Sm Y RNA，以及7种C/D盒小核仁RNA（snoRNA）。进一步分析表明，SL RNA和Sm Y RNA共享一个非常相似的Sm结合位点元件（AAU4 - 5GGA），其序列组成与其他U snRNA略有不同。沉淀物中还有16种没有Sm结合位点元件的ncRNA，这表明对于这些ncRNA，TMG的形成可能独立于Sm蛋白发生。
我们的结果表明，大多数预测由RNA聚合酶II转录的ncRNA具有TMG帽，而那些预测由RNA聚合酶III转录或位于内含子中的ncRNA则没有TMG帽结构。与没有TMG帽的ncRNA相比，有TMG帽的ncRNA往往具有更高的表达水平。5种功能未注释的ncRNA也具有TMG帽结构，这可能有助于确定这些ncRNA的细胞功能。
