Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana
Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana
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DOI：
10.3389/fpls.2019.01116
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发表时间：
2019-09-19
影响因子：
5.6
通讯作者：
Zhang, Xiao-Ning
中科院分区：
文献类型：
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作者：
Chen, Samuel L.;Rooney, Timothy J.;Zhang, Xiao-Ning
Pre-mRNA选择性剪接是真核细胞利用现有遗传资源创造多样化蛋白质产物的保守机制。它与RNA代谢中的其他事件协调调节，如转录、聚腺苷化、RNA转运和通过蛋白质网络的无义介导的衰变。SR45 （SERINE/ARGININE-RICH 45）被认为是一个中性剪接调节剂。它与凋亡和剪接相关蛋白（ASAP）复合体的一个组成部分同源，在多个水平上调节RNA代谢。在这种背景下，我们试图理解为什么sr45-1突变体拟南芥有畸形的花朵、延迟的开花时间和增加的抗病性。先前的研究表明，SR45 -1突变体中一些抗病基因和开花抑制基因开花位点C （FLC）的表达增加，并且SR45与生殖过程相关rna之间存在物理关联。本研究采用串联质谱法（Tandem Mass Tag-based quantitative Mass spectrometry）比较了野生型（Col-0）和sr45-1突变体植株的花序蛋白丰度。共有7206个蛋白被量化，其中227个蛋白的积累差异显著。这些蛋白质中只有一小部分与表达改变的rna数据集重叠。蛋白质组学结果显示，sr45-1突变体增加了硫代葡萄糖苷生物合成酶的数量，这对抗病至关重要。此外，突变花序的sin3相关蛋白18 （SAP18）的数量急剧减少，SAP18是第二种ASAP复合物成分，尽管SAP18 RNA没有显著减少。在sr45-1突变体中，第三个ASAP成分蛋白ACINUS的丰度也较低，但RNA没有明显变化。为了验证SR45对SAP18的影响，我们在转基因拟南芥Col-0和SR45 -1植株中过量产生了SAP18- gfp融合蛋白。在没有SR45的情况下，SAP18-GFP在细胞核（ASAP复合体的活性部位）的积累较少。此外，与非转基因sr45-1突变体相比，过量生产SAP18-GFP的转基因sr45-1突变体表达了更多的FLC，并且开花延迟更严重。这些结果表明，SR45是维持野生型细胞核中SAP18蛋白积累水平所必需的，而flc调控的开花时间是由ASAP复合体的正确表达和定位调控的。
Pre-mRNA alternative splicing is a conserved mechanism for eukaryotic cells to leverage existing genetic resources to create a diverse pool of protein products. It is regulated in coordination with other events in RNA metabolism such as transcription, polyadenylation, RNA transport, and nonsense-mediated decay via protein networks. SERINE/ARGININE-RICH 45 (SR45) is thought to be a neutral splicing regulator. It is orthologous to a component of the apoptosis and splicing-associated protein (ASAP) complex functioning to regulate RNA metabolism at multiple levels. Within this context, we try to understand why the sr45-1 mutant Arabidopsis has malformed flowers, delayed flowering time, and increased disease resistance. Prior studies revealed increased expression for some disease resistance genes and the flowering suppressor Flowering Locus C (FLC) in sr45-1 mutants and a physical association between SR45 and reproductive process-related RNAs. Here, we used Tandem Mass Tag-based quantitative mass spectrometry to compare the protein abundance from inflorescence between Arabidopsis wild-type (Col-0) and sr45-1 mutant plants. A total of 7,206 proteins were quantified, of which 227 proteins exhibited significantly different accumulation. Only a small percentage of these proteins overlapped with the dataset of RNAs with altered expression. The proteomics results revealed that the sr45-1 mutant had increased amounts of enzymes for glucosinolate biosynthesis which are important for disease resistance. Furthermore, the mutant inflorescence had a drastically reduced amount of the Sin3-associated protein 18 (SAP18), a second ASAP complex component, despite no significant reduction in SAP18 RNA. The third ASAP component protein, ACINUS, also had lower abundance without significant RNA changes in the sr45-1 mutant. To test the effect of SR45 on SAP18, a SAP18-GFP fusion protein was overproduced in transgenic Arabidopsis Col-0 and sr45-1 plants. SAP18-GFP has less accumulation in the nucleus, the site of activity for the ASAP complex, without SR45. Furthermore, transgenic sr45-1 mutants overproducing SAP18-GFP expressed even more FLC and had a more severe flowering delay than non-transgenic sr45-1 mutants. These results suggest that SR45 is required to maintain the wild-type level of SAP18 protein accumulation in the nucleus and that FLC-regulated flowering time is regulated by the correct expression and localization of the ASAP complex.