College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, PR China.
J Exp Bot. 2014 Feb;65(2):395-400. doi: 10.1093/jxb/ert409. Epub 2013 Dec 11.
In plants, Dicer-like 1 (DCL1)-mediated two-step cleavages are essential for the processing of microRNA (miRNA) gene products. Interestingly, DCL1 has been indicated to be involved in the production of many small RNAs (sRNAs) that cannot be classified as canonical miRNAs. However, genomic and functional information on the non-miRNA, DCL1-dependent sRNAs is still limited. Here, we propose a secondary structure-based approach for identification of the precursors containing novel DCL1-dependent sRNA loci. To demonstrate the utility of the workflow: first, 5898 DCL1-dependent sRNAs of 20-24 nucleotides were identified from the sRNA high-throughput sequencing data sets prepared from rice DCL1 RNA interference transgenic lines. Those perfectly mapped to the rice pre-miRNAs (precursor microRNAs) were removed. The remaining 5795 sRNAs were then mapped onto the rice genome, obtaining 30 902 perfectly matched loci belonging to 2310 sRNAs. A total of 4631 clusters of sRNA loci were defined for secondary structure prediction by using RNAfold. The prediction results generated by two algorithms, namely MFE (minimum free energy) and centroid, were manually compared to identify the conserved long-stem structures containing DCL1-dependent sRNA loci. For the purpose of a case study, a portion of the prediction results was screened manually. As a result, 60 clusters displayed great potential for forming featured long-stem structures for the generation of DCL1-dependent sRNAs. Together, the results indicate that the proposed workflow is applicable for the identification of novel DCL1-dependent sRNA loci on plant genomes.
在植物中，Dicer-like 1（DCL1）介导的两步切割对于 microRNA（miRNA）基因产物的加工至关重要。有趣的是，DCL1 已被表明参与许多不能归类为典型 miRNA 的小 RNA（sRNA）的产生。然而，关于非 miRNA、DCL1 依赖性 sRNA 的基因组和功能信息仍然有限。在这里，我们提出了一种基于二级结构的方法来鉴定包含新的 DCL1 依赖性 sRNA 基因座的前体。为了证明工作流程的实用性：首先，从水稻 DCL1 RNA 干扰转基因系的 sRNA 高通量测序数据集中鉴定了 5898 个 20-24 个核苷酸的 DCL1 依赖性 sRNA。那些完全映射到水稻前体 miRNA（前体 microRNA）的被去除。然后，将剩余的 5795 个 sRNA 映射到水稻基因组上，得到 30902 个完全匹配的基因座，属于 2310 个 sRNA。通过使用 RNAfold 对 30902 个完全匹配的基因座进行二级结构预测，共定义了 4631 个 sRNA 基因座簇。使用两种算法（最小自由能（MFE）和质心）对预测结果进行了手动比较，以鉴定包含 DCL1 依赖性 sRNA 基因座的保守长茎结构。为了进行案例研究，一部分预测结果被手动筛选。结果，60 个簇显示出形成特征长茎结构以产生 DCL1 依赖性 sRNA 的巨大潜力。总之，这些结果表明，所提出的工作流程适用于鉴定植物基因组上的新的 DCL1 依赖性 sRNA 基因座。
