RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA.
RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA.
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DOI：
10.1021/acs.biochem.2c00096
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发表时间：
2022-05-17
期刊：
影响因子：
2.9
通讯作者：
Cho, Samuel S.
中科院分区：
文献类型：
--
作者：
Koculi, Eda;Cho, Samuel S.
大肠杆菌50 S核糖体大亚基的23 S核糖体RNA（rRNA）含有26个转录后修饰的核苷。在这里，我们确定的35 S和45 S大亚基中间体的修饰程度，积累在细胞表达解旋酶失活DbpA蛋白，R331 A，和天然50 S大亚基。我们的特点是修改3-甲基假尿苷，2-甲基腺嘌呤，5-羟基胞苷，和9个假尿苷。用1-环己基-3-（2-吗啉代乙基）碳二亚胺甲基对甲苯磺酸盐（CMCT）处理，然后用碱处理，检测这些修饰。此外，23 S rRNA的高锰酸钾处理用于检测5-羟基胞苷修饰。CMCT和KMnO 4处理在修饰的核苷酸中产生化学变化，导致逆转录酶错配和缺失，这是使用下一代测序检测到的。我们的研究结果表明，2-甲基腺嘌呤修饰和7个尿苷假尿苷异构化存在于35 S和45 S中的50 S相似的程度。因此，进行这些修饰的酶，即RluA、RluB、RluC、RluE、RluF和RlmN，已经在中间体中起作用。与50 S相比，35 S和45 S中的两种尿苷到假尿苷异构化，即3-甲基假尿苷和5-羟基胞苷修饰，显著较少。因此，掺入这些修饰的酶RluD、RlmH和RlhA处于修饰35 S和45 S的过程中，或者将在核糖体组装的后期阶段掺入这些修饰。我们的研究采用了一种新的高通量和单核苷酸分辨率技术检测2-甲基腺嘌呤和两种新的高通量和单核苷酸分辨率技术检测5-羟基胞苷。
23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are 3-methylpseudouridine, 2-methyladenine, 5-hydroxycytidine, and nine pseudouridines. These modifications were detected using 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesul-fonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytidine modification. CMCT and KMnO4 treatments produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next-generation sequencing. Our results show that the 2-methyladenine modification and seven uridines to pseudouridine isomerizations are present in both the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely, RluA, RluB, RluC, RluE, RluF, and RlmN, have already acted in the intermediates. Two uridines to pseudouridine isomerizations, the 3-methylpseudouridine and 5-hydroxycytidine modifications, are significantly less present in the 35S and 45S, as compared to the 50S. Therefore, the enzymes that incorporate these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S and 45S or will incorporate these modifications during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for the detection of 2-methyladenine and two novel high throughput and single nucleotide resolution techniques for the detection of 5-hydroxycytidine.
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14.9
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