Department of Horticulture and Department of Forestry, Michigan State University, East Lansing, MI, 48824-1222, USA; DOE-Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI, 48824-1222, USA.
Plant J. 2014 Dec;80(5):772-84. doi: 10.1111/tpj.12667. Epub 2014 Nov 7.
AtC3H14 (At1 g66810) is a plant-specific tandem CCCH zinc-finger (TZF) protein that belongs to the 68-member CCCH family in Arabidopsis thaliana. In animals, TZFs have been shown to bind and recruit target mRNAs to the cytoplasmic foci where mRNA decay enzymes are active. However, it is not known whether plant TZF proteins such as AtC3H14 function. So far, no mRNA targets of plant TZFs have been identified. We have obtained several lines of experimental evidence in support of our hypothesis that AtC3H14 is involved in post-transcriptional regulation of its target genes. Nucleic acid binding assays using [(35) S]-labeled AtC3H14 protein showed that AtC3H14 could bind to ssDNA, dsDNA, and ribohomopolymers, suggesting its RNA-binding activity. RNA immunoprecipitation (RIP) assay identified several putative target RNAs of AtC3H14, including a polygalacturonase, a well-known cell wall modifying gene. RNA electrophoretic mobility shift assays (RNA-EMSA) were used to confirm the RIP results and demonstrate that the TZF domain of AtC3H14 is required for the target RNA binding. Microarray analysis of 35S::AtC3H14 plants revealed that many of the cell wall elongation and/or modification-associated genes were differentially expressed, which is consistent with the cell elongation defect phenotype and the changes in the cell wall monosaccharide composition. In addition, yeast activation assay showed that AtC3H14 also function as a transcriptional activator, which is consistent with the previous finding that AtC3H14 activate the secondary wall biosynthesis genes. Taken together, we conclude that AtC3H14 may play a key role in both transcriptional and post-transcriptional regulation.
AtC3H14（At1g66810）是一种植物特异性串联 CCCH 锌指（TZF）蛋白，属于拟南芥中 68 个成员的 CCCH 家族。在动物中，已经表明 TZF 可以结合并募集靶 mRNA 到细胞质焦点，其中 mRNA 降解酶是活跃的。然而，尚不清楚植物 TZF 蛋白（如 AtC3H14）是否发挥作用。到目前为止，尚未鉴定出植物 TZF 的 mRNA 靶标。我们已经获得了一些实验证据支持我们的假设，即 AtC3H14 参与其靶基因的转录后调控。使用 [(35)S]-标记的 AtC3H14 蛋白进行核酸结合测定表明，AtC3H14 可以与 ssDNA、dsDNA 和核糖同聚物结合，表明其 RNA 结合活性。RNA 免疫沉淀（RIP）测定鉴定了 AtC3H14 的几个假定靶 RNA，包括一种多聚半乳糖醛酸酶，一种已知的细胞壁修饰基因。RNA 电泳迁移率变动分析（RNA-EMSA）用于确认 RIP 结果并证明 AtC3H14 的 TZF 结构域是靶 RNA 结合所必需的。35S::AtC3H14 植物的微阵列分析显示，许多细胞壁伸长和/或修饰相关基因表达差异，这与细胞伸长缺陷表型和细胞壁单糖组成的变化一致。此外，酵母激活测定表明 AtC3H14 也作为转录激活因子发挥作用，这与之前发现 AtC3H14 激活次生壁生物合成基因的结果一致。总之，我们得出结论，AtC3H14 可能在转录和转录后调控中都发挥关键作用。
