College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, China.
Weifang University of Science and Technology, Weifang, 262700, China.
Plant Cell Rep. 2024 Oct 5;43(10):252. doi: 10.1007/s00299-024-03340-6.
The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein-protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling.
拟南芥 RNA 解旋酶 LOS4 在调控乙烯信号通路中 EIN2、CTR1 和 ERS2 等基因的前体 mRNA 剪接中起着关键作用。植物激素乙烯在植物生长、发育和应激反应中发挥着多种作用。乙烯通过膜结合的乙烯受体复合物感知，然后触发下游成分，如 EIN2，启动信号转导进入细胞核，导致乙烯应答基因的激活。在过去的几十年中，关于乙烯途径中的基因克隆、蛋白-蛋白相互作用和下游基因表达，已经积累了大量的信息。然而，我们对乙烯信号通路中关键基因的 mRNA 转录后加工和修饰的理解仍然有限。本研究旨在提供证据，证明拟南芥 RNA 解旋酶 LOS4 参与乙烯信号通路中 EIN2、CTR1 和 ERS2 基因的前体 mRNA 剪接。本研究采用了 RNAi 基因沉默、CRISPR-Cas9 基因编辑和氨基酸突变等各种遗传方法。当 LOS4 被沉默或敲除时，黄化幼苗对乙烯的敏感性显著增强。进一步的研究表明，当 LOS4 被敲除时，EIN2 前体 mRNA 的剪接出现错误。此外，在该途径中的 ERS2 和 CTR1 基因中观察到异常的前体 mRNA 剪接。生化分析表明，los4-2（E94K）突变蛋白表现出增强的 ATP 结合和增强的 ATP 水解活性。相反，los4-1（G364R）突变体的底物 RNA 结合减少，ATP 结合活性降低。这些发现显著提高了我们对 RNA 解旋酶在乙烯信号转导中的调控功能和分子机制的理解。
