Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry
Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry
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DOI：
10.1017/s1355838200992148
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发表时间：
2000-02-01
期刊：
影响因子：
4.5
通讯作者：
Roepstorff, P
中科院分区：
文献类型：
--
作者：
Kirpekar, F;Douthwaite, S;Roepstorff, P
我们提出了一种基于基质辅助激光解吸/电离质谱仪(MALDI-MS)筛选RNA转录后修饰的方法。在RNA被核苷酸特异性核糖核酸酶消化完成后，对片段进行质谱分析。将观察到的大量数据与从基因序列预测的数据进行比较，确定含有修饰核苷酸的片段。比二核苷酸大的片段对识别转录后修饰很有价值。通过并行使用两个RNase并结合源后衰减(PSD)的进一步片段，可以获得更精细的RNA修饰图谱。该方法可以快速、灵敏地筛选出用于转录后修饰的纯化RNA，并已应用于两种嗜热微生物--脂肪嗜热芽孢杆菌和古生菌Sulfolobus acidocaldarius以及嗜盐古菌Halbacterium halobium和Haloarcula marismortui的5S rRNA上。鉴定出一种嗜酸性链球菌转录后修饰，并进一步用PSD鉴定为胞苷(32)的甲基化。修饰后的C位于明显保守的区域，该区域在嗜热脂肪杆菌和嗜盐梭菌中都是明显保守的，在一定程度上在海洋嗜热杆菌中也是如此。然而，在后三种生物中没有发现类似的修饰。我们进一步发现，与所研究的其他5S rRNA物种相反，盐生H.halobium 5S rRNA的5‘端被去磷酸化。该方法还提供了预期的RNA序列是否与观察到的片段质量一致的即时指示。与已发表的两个5S rRNA序列存在差异，并在此报告。
We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/ Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine(32). The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.