Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7.
Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7.
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DOI：
10.1186/s13059-017-1332-x
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发表时间：
2017-10-31
期刊：
影响因子：
12.3
通讯作者：
Staiger D
中科院分区：
文献类型：
--
作者：
Meyer K;Köster T;Nolte C;Weinholdt C;Lewinski M;Grosse I;Staiger D
rna结合蛋白在调控植物发育和环境反应中的作用已经得到了很好的证实。然而，缺乏其体内结合靶点和结合景观的全基因组视图代表了理解植物rna结合蛋白的作用模式的空白。在这里，我们采用个体核苷酸分辨率交联和免疫沉淀（iCLIP）全基因组来确定生物钟调节的拟南芥富含甘氨酸的rna结合蛋白AtGRP7的结合库。iCLIP在表达AtGRP7-GFP的植物中鉴定出858个交联位点显著富集的转录本，而这些转录本在表达rna结合死亡的AtGRP7变体或单独表达GFP的植物中则不存在。为了独立验证靶标，我们对甲醛固定的AtGRP7-GFP植物进行了RNA免疫沉淀（RIP）测序。在iCLIP靶点中，452个也通过RIP-seq鉴定，代表了一组高置信度的结合物。AtGRP7可以结合所有转录区，优先结合3 '非翻译区。在交联位点附近，富U/ c基序被过多地代表。交叉对照AtGRP7功能缺失突变体或AtGRP7过表达植物中转录组变化的靶标，揭示了AtGRP7对其靶标的主要负面影响。特别是，AtGRP7水平的升高导致转录本昼夜振荡的抑制，包括休眠/生长素相关家族蛋白2和ccr样蛋白。此外，随着AtGRP7水平的改变，一些靶标显示出选择性剪接或聚腺苷化的变化。我们已经为植物建立了iCLIP来鉴定rna结合蛋白AtGRP7的目标转录本。这为研究响应外源性和内源性线索的转录后网络动力学铺平了道路。本文的在线版本（doi:10.1186/s13059-017-1332-x）包含补充材料，仅供授权用户使用。
Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. iCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3′ untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels. We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues. The online version of this article (doi:10.1186/s13059-017-1332-x) contains supplementary material, which is available to authorized users.
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