Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA.
Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA.
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DOI：
10.1021/acs.biochem.7b00323
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发表时间：
2017-09-05
期刊：
影响因子：
2.9
通讯作者：
Simon MD
中科院分区：
文献类型：
--
作者：
Sexton AN;Wang PY;Rutenberg-Schoenberg M;Simon MD
化学探针有能力提供对体内和体外RNA构象的洞察，但解释结果取决于检测化学修饰核苷酸的方法。传统上，修饰碱基的存在是通过它们在引物延伸过程中阻止逆转录酶的能力和电泳或测序观察到的终止位点的位置来推断的。最近，修饰诱导突变已被用作化学探测数据的读数。考虑到不同逆转录酶对错配结合和通读的不同倾向，我们研究了在相同的化学探测实验中，终止和突变事件如何相互比较。我们发现由硫酸二甲酯探针诱导的突变和终止都是针对甲基化碱基的，但这两种测量结果的相关性令人惊讶地小，并且在很大程度上代表了化学修饰数据的非重叠指标。我们还表明，对修饰碱基的特定偏差部分取决于局部序列背景，不同的逆转录酶在将修饰视为停止或突变方面表现出不同的偏差。这些结果支持将终止和突变事件分析纳入RNA探测实验的方法。
Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.
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