Gualtieri Carla, Gianella Maraeva, Pagano Andrea, Cadeddu Tiziano, Araújo Susana, Balestrazzi Alma, Macovei Anca
Plant Biotechnology Laboratory, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
Front Plant Sci. 2021 Mar 9;12:645323. doi: 10.3389/fpls.2021.645323. eCollection 2021.
One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an system was developed, using the model legume . Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants.
生物体面临的挑战之一是迅速应对基因毒性应激以避免DNA损伤。为此，包括植物在内的所有生物体都进化出了复杂的DNA损伤反应（DDR）机制。这些机制在生物体中高度保守，需要精细调控。在这种情况下，微小RNA（miRNA）正成为活跃的参与者，从而吸引了研究界的关注。miRNA在DDR中的作用在人类细胞中得到了广泛研究，而在植物中的研究仍然很少。为了通过实验研究植物miRNA在DDR相关途径调控中的作用，利用模式豆科植物开发了一个系统。使用喜树碱（CPT）和/或NSC120686（NSC）进行特定处理，分别靶向DDR的不同组分，即拓扑异构酶I（TopI）和酪氨酰-DNA磷酸二酯酶1（TDP1）。表型（发芽率和速度、幼苗生长）和分子（细胞死亡、DNA损伤和基因表达谱）分析表明，施加的处理会影响DDR。我们的结果表明，这些处理不会影响发芽过程，而是抑制幼苗发育，导致细胞死亡增加和DNA损伤积累。此外，还观察到植物DDR的主调节因子γ反应抑制因子1（）表达的处理特异性变化。此外，在不同DNA修复途径和细胞周期调控中起重要作用的多个基因的表达以处理特异性方式差异表达。随后，研究了我们之前通过生物信息学方法鉴定的可能靶向DDR过程相关基因的特定miRNA及其靶标。获得的结果表明，在大多数情况下，当miRNA上调时，相应的候选靶基因下调，这为miRNA对这些靶标的作用提供了间接证据。因此，本研究扩展了我们目前关于miRNA在植物DDR转录后调控中作用的现有知识。
