Wang Changlin, Washida Haruhiko, Crofts Andrew J, Hamada Shigeki, Katsube-Tanaka Tomoyuki, Kim Dongwook, Choi Sang-Bong, Modi Mahendra, Singh Salvinder, Okita Thomas W
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Plant J. 2008 Aug;55(3):443-54. doi: 10.1111/j.1365-313X.2008.03516.x. Epub 2008 Apr 12.
Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.
先前的研究表明，水稻胚乳中发现的主要储存蛋白RNA通过肌动球蛋白作为颗粒运输到皮质内质网的特定亚结构域。在本研究中，我们研究了主要的细胞骨架相关RNA结合蛋白OsTudor-SN在RNA运输和定位中的潜在作用。OsTudor-SN分子以高分子量形式存在，其完整性对RNase敏感。免疫沉淀后进行RT-PCR表明OsTudor-SN结合醇溶蛋白和谷蛋白RNA。使用亲和纯化抗体的免疫荧光研究表明，OsTudor-SN以颗粒形式存在于细胞质中，并分布到蛋白体内质网（ER）和潴泡内质网。在表达GFP标记的醇溶蛋白RNA运输颗粒的转基因水稻植株中检查OsTudor-SN颗粒，结果显示OsTudor-SN和GFP共定位，表明其在RNA运输中起作用。与此观点一致，在活的胚乳切片中观察到GFP标记的OsTudor-SN作为移动颗粒，这种特性受到微丝抑制剂的抑制。通过反义和RNAi下调OsTudor-SN导致醇溶蛋白RNA和蛋白质的稳态水平降低，以及醇溶蛋白蛋白体数量减少。总体而言，这些结果表明OsTudor-SN是RNA运输颗粒的一个组成部分，并且可能通过调节导致其RNA运输、定位和锚定到皮质内质网的一个或多个过程来控制储存蛋白的生物合成。
