Köster Tino, Meyer Katja, Weinholdt Claus, Smith Lisa M, Lummer Martina, Speth Corinna, Grosse Ivo, Weigel Detlef, Staiger Dorothee
Molecular Cell Physiology, Bielefeld University.
Institute of Computer Science, Martin-Luther-University Halle-Wittenberg, Germany.
Nucleic Acids Res. 2014 Sep;42(15):9925-36. doi: 10.1093/nar/gku716. Epub 2014 Aug 7.
The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.
类hnRNP富含甘氨酸的RNA结合蛋白AtGRP7调控拟南芥中的前体mRNA剪接。在此,我们利用小RNA测序表明AtGRP7也会影响miRNA库。AtGRP7的过表达导致30种miRNA的水平显著降低,14种miRNA的水平升高,最小log2倍数变化为±0.5。包括pri-miR398b、pri-miR398c、pri-miR172b、pri-miR159a和pri-miR390在内的几种初级miRNA过度积累,而成熟miRNA的水平则相应降低,这表明AtGRP7影响初级miRNA的加工。事实上,RNA免疫沉淀显示AtGRP7在体内与这些初级miRNA相互作用。RNA识别基序中一个精氨酸的突变消除了体内结合以及对miRNA和初级miRNA水平的影响,表明AtGRP7通过直接结合抑制这些初级miRNA的加工。相比之下,在高AtGRP7水平下升高或未发生变化的特定miRNA的初级miRNA在体内未被结合。miR390(反式作用小干扰RNA(ta-siRNA)形成的起始物)积累的减少也导致TAS3 ta-siRNA水平降低以及靶标生长素响应因子4的mRNA表达增加。此外,AtGRP7影响pri-miR172b和pri-miR162a的剪接。因此,AtGRP7是一种类hnRNP蛋白,除了在前体mRNA剪接中的作用外,还在初级miRNA的加工中发挥作用。