School of Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia.
PLoS One. 2012;7(4):e35933. doi: 10.1371/journal.pone.0035933. Epub 2012 Apr 24.
The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).
Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.
拟南芥（Arabidopsis）双链 RNA 结合（DRB）蛋白家族由五个成员组成，分别是 DRB1 到 DRB5。两种重要的发育小分子 RNA（sRNA）物种，microRNAs（miRNAs）和转座小干扰 RNA（tasiRNAs）的生物发生需要 DICER-LIKE（DCL）内切酶分别辅助 DRB1 和 DRB4。miRNA 指导的靶基因表达在植物发育中的重要性体现在 DRB1 活性丧失（drb1 植物）的表型后果中。
在这里，我们报告说，drb235 三重突变体植物的发育表型是 shoot apical meristem（SAM）区域 miRNA 生物发生失调的结果。DRB2、DRB3 和 DRB5 在野生型幼苗中的表达仅限于 SAM 区域。drb235 植物相应组织的小 RNA 测序显示 miRNA 积累发生改变。大约一半检测到的 miRNAs 水平与野生型植物相当。然而，在三重突变体中，其余 miRNAs 的积累要么升高，要么降低。对不同的单突变体和多重突变体的检查表明，DRB2 活性丧失与升高和降低的 miRNA 类别的积累改变之间存在明显的关联。此外，我们表明，DRB2 在其野生型表达域之外的组成型过表达可以补偿 drb1 植物中 DRB1 活性的丧失。
结论/意义：我们的结果表明，在 SAM 区域，DRB2 既拮抗又协同 DRB1 在 miRNA 生物发生中的作用，在这个发育重要的组织中增加了一层额外的基因调控复杂性。
