Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.
Anomalous Reverse Transcription through Chemical Modifications in Polyadenosine Stretches.
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DOI：
10.1021/acs.biochem.0c00020
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发表时间：
2020-06-16
期刊：
影响因子：
2.9
通讯作者：
Das R
中科院分区：
文献类型：
--
作者：
Kladwang W;Topkar VV;Liu B;Rangan R;Hodges TL;Keane SC;Al-Hashimi H;Das R
耐热逆转录酶是几乎所有RNA结构定位和转录组范围内RNA化学修饰发现的现代技术的基础。尽管它们被广泛使用，但这些酶在化学修饰核苷酸上的行为仍然知之甚少。Wellington-Oguri等人最近报道，通过逆转录检测，经硫酸二甲酯或2′羟基酰化修饰的推定非结构化聚腺苷延伸段中化学修饰的明显缺失。在这里，对这些和其他公开可用数据的重新分析，对化学修饰rna的毛细管电泳实验，以及对A12及其变体的核磁共振波谱分析表明，这种影响不太可能是由聚腺苷的异常结构引起的。相反，不同的逆转录酶对化学修饰的rna和由单个1-甲基腺苷合成的分子的测试暗示了一种以前未被表征的逆转录酶行为：通过多腺苷延伸的化学修饰进行近定量的旁路。所有被测试的天然和工程逆转录酶（MMLV、SuperScript II、III和IV、tgrt -III和MarathonRT）都表现出这种异常的旁路行为。精确的dms引导的多腺苷化hiv - 13 '非翻译区RNA的结构建模需要考虑到这种异常。我们的研究结果表明，poly（rA-dT）杂化双链可以触发意想不到的有效逆转录酶旁路，并且poly(A) mRNA尾部的化学修饰通常可能被低估。
Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes’ behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2’ hydroxyl acylation, as probed by reverse transcription. Here, re-analysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on A12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3´ untranslated region RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.
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影响因子：
3.7
作者：
Coutsinos, Dimitrios;Invernizzi, Cedric F.;Wainberg, Mark A.
通讯作者：
Wainberg, Mark A.
影响因子：
14.9
作者：
Hauenschild R;Tserovski L;Schmid K;Thüring K;Winz ML;Sharma S;Entian KD;Wacheul L;Lafontaine DL;Anderson J;Alfonzo J;Hildebrandt A;Jäschke A;Motorin Y;Helm M
通讯作者：
Helm M
影响因子：
2.9
作者：
Kladwang W;Mann TH;Becka A;Tian S;Kim H;Yoon S;Das R
通讯作者：
Das R
影响因子：
14.9
作者：
Eichhorn CD;Feng J;Suddala KC;Walter NG;Brooks CL 3rd;Al-Hashimi HM
通讯作者：
Al-Hashimi HM
DOI：
10.1261/rna.065219.117
发表时间：
2018-03
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Darnell RB;Ke S;Darnell JE Jr
通讯作者：
Darnell JE Jr