Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends.
Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends.
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DOI：
10.3389/fpls.2021.793549
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发表时间：
2021
影响因子：
5.6
通讯作者：
Ren G
中科院分区：
文献类型：
--
作者：
Li N;Ren G
在植物中，RNase III型酶Dicer-like 1（DCL 1）从其初级转录物（称为pri-miRNAs）中加工大多数microRNA（miRNAs）。四种不同的处理模式（即，短碱基到环、顺序碱基到环、短环到碱基和顺序环到碱基）的特征，主要通过RNA末端的特异性平行扩增（SPARE）方法。然而，SPARE是一种靶向克隆方法，需要优化每个靶点的克隆效率和特异性。帕雷（RNA末端的平行扩增）本身是一种非靶向方法，广泛用于鉴定miRNA介导的靶切割事件。帕雷在表征miRNA加工模式中的主要问题是成熟miRNA的潜在污染。在这里，我们提供了一种估计miRNA污染水平的方法，并表明大多数公开可用的帕雷文库具有可忽略的miRNA污染。帕雷检测到的拟南芥中的蛋白质数目和加工方式与SPARE检测到的相似。帕雷还确定了36个拟南芥miRNAs的加工模式，这是SPARE未探索的，表明它可以补充SPARE方法。利用公开的帕雷数据集，我们分别鉴定了玉米、水稻、大豆和番茄中36、91、90和54种miRNA的加工模式，并证明了每个miRNA家族中的加工模式总体上是保守的。通过追踪miRNA加工残留物的能力，帕雷还促进了miRNA的表征和注释。
In plants, the RNase III-type enzyme Dicer-like 1 (DCL1) processes most microRNAs (miRNAs) from their primary transcripts called pri-miRNAs. Four distinct processing modes (i.e., short base to loop, sequential base to loop, short loop to base, and sequential loop to base) have been characterized in Arabidopsis, mainly by the Specific Parallel Amplification of RNA Ends (SPARE) approach. However, SPARE is a targeted cloning method which requires optimization of cloning efficiency and specificity for each target. PARE (Parallel Amplification of RNA Ends) is an untargeted method per se and is widely used to identify miRNA mediated target slicing events. A major concern with PARE in characterizing miRNA processing modes is the potential contamination of mature miRNAs. Here, we provide a method to estimate miRNA contamination levels and showed that most publicly available PARE libraries have negligible miRNA contamination. Both the numbers and processing modes detected by PARE were similar to those identified by SPARE in Arabidopsis. PARE also determined the processing modes of 36 Arabidopsis miRNAs that were unexplored by SPARE, suggesting that it can complement the SPARE approach. Using publicly available PARE datasets, we identified the processing modes of 36, 91, 90, and 54 miRNAs in maize, rice, soybean, and tomato, respectively, and demonstrated that the processing mode was conserved overall within each miRNA family. Through its power of tracking miRNA processing remnants, PARE also facilitated miRNA characterization and annotation.
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