Hua Xia, Berkowitz Nathan D, Willmann Matthew R, Yu Xiang, Lyons Eric, Gregory Brian D
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Genomics and Computational Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Noncoding RNA. 2021 Apr 27;7(2):28. doi: 10.3390/ncrna7020028.
RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20-24 nucleotide (nt) small RNAs (smRNAs). In , smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3-5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in and mutant plants. Correspondingly, plants that lack these proteins ( and mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.
RNA沉默途径以序列特异性方式在转录水平或转录后水平控制真核基因表达。在RNA沉默中，双链RNA（dsRNA）的产生会产生各种类型的20 - 24个核苷酸（nt）的小RNA（smRNA）。在植物中，smRNA通常来源于由六种基因组编码的RNA依赖性RNA聚合酶（RDR）蛋白之一合成的长dsRNA分子。然而，RDR依赖性smRNA的完整组成以及这些蛋白及其RNA结合辅因子在植物RNA沉默中所起的作用尚未完全揭示。为了填补这一空白，我们对所有六种RDR及其两种辅因子进行了全基因组分析，以寻找RDR的新底物以及由此产生的RDR衍生的siRNA的靶标，从而揭示这些蛋白在植物中的新功能。基于这些分析，我们鉴定出了三种RDRγ进化枝蛋白（RDR3 - 5）的底物，这些底物之前尚未得到充分表征。我们还鉴定出了其他三种RDR（RDR1、RDR2和RDR6）以及RDR2辅因子RNA指导的DNA甲基化12（RDM12）和RDR6辅因子基因沉默抑制因子3（SGS3）的新底物。这些发现表明，SGS3的靶标底物不仅限于RDR6单独利用的那些底物，而且该蛋白似乎是RDR蛋白家族更普遍的辅因子。此外，我们发现RDR6和SGS3参与了靶向与非生物胁迫相关转录本的smRNA的产生，这些胁迫包括缺水、盐胁迫和ABA反应，并且正如预期的那样，这些mRNA的水平在RDR6和SGS3突变植物中有所增加。相应地，与野生型植物相比，缺乏这些蛋白的植物（RDR6和SGS3突变体）对ABA处理高度敏感，对高浓度PEG8000具有耐受性，并且在盐处理下具有更高的存活率。总的来说，我们的分析为揭示植物中RDR依赖性RNA沉默的新功能提供了极其丰富的数据资源，同时也揭示了RDR6 / SGS3依赖性途径与植物非生物胁迫反应之间以前未被探索的联系。
