| 摘要: | 從單細胞酵母菌一直到多細胞動物，如：老鼠、人類等真核生物中都具有CCR4-NOT複合體，這個複合體對於基因表達是整體的調節者。複合體中的各個蛋白質對於不同層面的mRNA代謝都有關聯，包括transcription initiation、elongation 和mRNA degradation。控制mRNA的半衰期是調控基因表現的重要一環，大部分的情況下，調控真核細胞mRNA半衰期的主要途徑是先將mRNA的poly (A) tail縮短。在酵母菌的研究得知，CCR4蛋白質是CCR4-NOT複合體的其中一個成員，並已經證實在細胞質中具有deadenylation的功能。然而，水稻CCR4的功能目前尚未清楚，因此本篇論文將進一步探討CCR4在水稻中的功能。
透過胺基酸序列比對分析發現兩個水稻CCR4 homolog，並命名為OsCCR4a和OsCCR4b。水稻CCR4的C端nuclease domain重組蛋白質：His-OsCCR4a EEP 和His-OsCCR4b EEP，在in vitro的實驗中證實具有deadeanylase的酵素活性，在水稻CCR4的activity site點突變會使其deadeanylase的酵素活性消失。processing bodies是存在真核細胞的細胞質中顆粒狀結構，裡面包含了很多跟mRNA turnover有關的酵素，我們發現水稻CCR4會在細胞質的processing bodies中。基因表現的部分，發現OsCCR4a和OsCCR4b基因在葉子和劍葉的部分表現量比較高，並且會受到熱逆境所誘導，但是在賀爾蒙2,4D以及NAA的處理下，OsCCCR4b會被誘導而OsCCR4a的基因表現不會受到影響。兩個OsCCR4蛋白質都具有deadenylase的酵素活性，並且會在細胞質的processing bodies中，推測OsCCR4蛋白質可能參與水稻中 mRNA的deadeylation；從基因表現的部分，發現在水稻發育過程或受到環境壓力下OsCCR4a和OsCCR4b並不是完全的functional redundancy。
;The CCR4-NOT (carbon catabolite repression, negative on TATA-less) complex is a global regulator of gene expression that is conserved from yeast to human. The complex is involved in several aspects of mRNA metabolism, including transcription initiation, elongation and mRNA degradation. Control of mRNA turnover is a critical mechanism for regulating of gene expression in eukaryotice cells. One of major pathway of mRNA turnover in eukaryotic cells is initiated by mRNA deadenylation (also called as poly(A) tail shortening). In yeast, the CCR4 protein, as part of the CCR4-NOT complex, has been shown that take a responsibility for cytoplasmic deadenylation. However, the function of CCR4 in rice is not clear yet. In this study, we identified and cloned two CCR4 homologs, OsCCR4a and OsCCR4b, from Oryza sativa. The OsCCR4s C-terminal nuclease domain recombinant proteins, His-OsCCR4a EEP and His-OsCCR4b EEP, exhibited deadenylase activity in vitro, and mutation in their activity site abolished their deadenylase activities, respectively. Subcellular localization analysis showed that OsCCR4a-GFP and OsCCR4b-GFP are localized in cytoplasmic mRNA processing bodies (P-bodies), which are specific granules consisting of enzymes involved in mRNA turnover. Both OsCCR4s were suggested are heat stress related genes while their expression were induced by heat stress in rice. However, only OsCCR4b was induced by 2,4D and NAA treatments. Our findings provide biochemical evidence that two OsCCR4 proteins may be involved in the deadenylation in rice. The various expression patterns between two OsCCR4s implied that OsCCR4a and OsCCR4b aren’t completely functional redundancy in the developmental process and stress response of the rice. |
