Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19848-53. doi: 10.1073/pnas.0910754106. Epub 2009 Nov 10.
Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ( approximately 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed <<30% colocalization. mPP association with the peroxisome or ER was verified using cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the cell cycle, with its localization depending in part on the 3'-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.
靶向 mRNA 运输和局部翻译可能在控制蛋白质定位中发挥重要作用。在这里，我们首次检查了参与过氧化物酶体生物发生和功能的所有（约 50 个）编码过氧化物酶体蛋白（mPP）的 mRNAs 的定位。通过使用融合了多个 GFP 拷贝的噬菌体 MS2-CP RNA 结合蛋白（RBP），我们证明了 >40 个内源性表达的标记有 MS2 适体的 mPP 在体内形成荧光 RNA 颗粒。使用不同的 RFP 标记的细胞器标记物揭示了 mPP 颗粒定位的 3 种基本模式：到过氧化物酶体、内质网（ER）和非过氧化物酶体。12 个 mPP（即 PEX1、PEX5、PEX8、PEX11-15、DCI1、NPY1、PCS60 和 POX1）有高比例（52%-80%）的 mPP mRNA 与过氧化物酶体共定位。13 个 mPP（即 AAT2、PEX6、MDH3、PEX28 等）显示低比例（30%-42%）的共定位，1 个 mPP（PEX3）优先定位于 ER。非过氧化物酶体模式的 mPP（即 GPD1、PCD1、PEX7）显示 <<30% 的共定位。使用细胞分级分离和 RT-PCR 分析验证了 mPP 与过氧化物体或 ER 的关联。发现模型 mPP PEX14 mRNA 与整个细胞周期中的过氧化物体密切相关，其定位部分取决于 3'-UTR、翻译起始和 Puf5 RBP。观察到的 mPP 定位的不同模式表明，参与 mRNA 定位和翻译的多种机制可能在蛋白质导入过氧化物体中发挥作用。
