Department of Gene Expression, Faculty of Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University Poznan, Poland.
Front Plant Sci. 2012 Mar 19;3:46. doi: 10.3389/fpls.2012.00046. eCollection 2012.
Arabidopsis miR319a/b/c primary transcripts are unusual due to the presence of a long stem and loop structure containing functional miR319a/b/c molecules. In our experiments carried out using high throughput sequencing (HTS), we have shown that additional microRNAs (miRNAs), miR319a.2/b.2/c.2 are generated from the upper part of the same hairpin structure. We have also found cognate miRNAa.2*/b.2*/c.2* to be present in the HTS results with a considerably lower number of reads. Northern hybridization revealed that miR319b.2 is mainly expressed in 35-day-old plant rosette leaves, as well as in stem and inflorescences of 42- and 53-day-old plants. Moreover, it carries multiple signatures of a functional miRNA, including as follows: (i) its biogenesis is HYL1-dependent; (ii) it is incorporated in a substantial amount into RISC complexes containing AGO1, AGO2, or AGO4 protein; (iii) 24 nt-long species of miR319b.2 have been found in inflorescences to be more abundant than 21 nt miR319b.2 species; (iv) it is present in various ratios to miR319b during plant development, which suggests the existence of a regulatory mechanism responsible for its biogenesis/processing; (v) there is an observed cross-species conservation of the miR319a/b/c stem nucleotide sequence extending beyond mature miRNA region; and (vi) all evidence suggests that intron-containing RAP2.12 mRNA isoform is the target for miR319b.2. All these features prompt us to claim miR319b.2 as a functional miRNA molecule.
拟南芥 miR319a/b/c 初级转录本因其存在长茎和环结构而不同，该结构包含功能性 miR319a/b/c 分子。在我们使用高通量测序（HTS）进行的实验中，我们表明，额外的 microRNAs (miRNAs)，miR319a.2/b.2/c.2 是从同一发夹结构的上部产生的。我们还发现，在 HTS 结果中存在同源 miRNAa.2*/b.2*/c.2*，但其读数要少得多。Northern 杂交显示，miR319b.2 主要在 35 天大的植物莲座叶中表达，以及在 42 天和 53 天大的植物的茎和花序中表达。此外，它带有多个功能性 miRNA 的特征，包括：(i) 其生物发生依赖于 HYL1；(ii) 它以大量的形式掺入包含 AGO1、AGO2 或 AGO4 蛋白的 RISC 复合物中；(iii) 在花序中发现 24 个核苷酸长的 miR319b.2 物种比 21 个核苷酸长的 miR319b.2 物种更丰富；(iv) 它在植物发育过程中与 miR319b 以各种比例存在，这表明存在负责其生物发生/加工的调节机制；(v) 在成熟 miRNA 区域之外，观察到 miR319a/b/c 茎核苷酸序列的跨物种保守性；以及 (vi) 所有证据都表明，内含子的 RAP2.12 mRNA 同工型是 miR319b.2 的靶标。所有这些特征促使我们声称 miR319b.2 是一种功能性 miRNA 分子。
