臺灣博碩士論文加值系統

DDX3屬於DEAD box RNA 解旋酶的一員。DEAD box蛋白在RNA processing過程中，從上游的轉錄、pre-mRNA剪輯、mRNA出核，到下游的轉譯以及mRNA的降解 (degradation)，均扮演重要角色。在本實驗室先前的研究結果顯示DDX3可調控p21waf1/cif1基因啟動子的活化，並且也發現DDX3會與一些mRNA剪輯因子有交互作用。同時當DDX3存在細胞質中時，會與轉譯起始因子eIF4E作用，抑制cap-dependent的蛋白起始轉譯，顯示出DDX3是一個多功能的RNA解旋酶。此外，在Yedavalli等人的研究中，發現DDX3可分別與CRM1 (export receptor)及Rev (adaptor)蛋白有交互作用，並參與在HIV 的Rev-CRM1蛋白複合物將其未剪輯或部分剪輯的轉錄片段送出核的過程中。基於上述之研究報告，本論文欲探討DDX3在運送細胞的mRNA出核過程中所扮演的角色。
已知在CRM1-dependent mRNA export路徑中，HuR扮演adaptor蛋白，負責運送富含AU序列的mRNAs出核，因此，首先想確認DDX3是否存在於含有HuR-CRM1的mRNP中。藉由活體內共同免疫沉澱的方式，證實DDX3會與HuR交互作用，同時在heat shock處理後，此交互作用會顯著增加。而在免疫螢光染色的實驗中，同樣也可觀察到heat shock處理後，DDX3會與HuR共同座落在特殊的細胞質foci中。因此，欲進一步檢視DDX3是否會和特定種類，例如富含AU序列的mRNAs結合。藉由免疫沉澱與Flag-tagged DDX3交互作用之mRNP複合體，抽取免疫沉澱物中的RNAs進行RT-PCR，發現DDX3蛋白在正常情況下，會與DDX3 mRNA結合，其中也包含DDX3內生性轉錄片段(endogenous transcripts)。然而在heat shock處理過後，DDX3會與一些已知的HuR ligands結合，如c-fos、c-myc以及p21 mRNAs，這些mRNAs的3’UTR皆富含AU序列。另一方面，在DDX3 knockdown的HeLa細胞中，利用mRNA螢光原位雜交的實驗，觀察CRM1-dependent mRNA c-fos的出核情形，發現DDX3 表現量下降對於c-fos mRNA在細胞核與細胞質中的分布並無顯著改變。
此外，本論文也對於細胞在遭受如heat shock的環境壓力時，DDX3在細胞質中聚集形成的foci做了進一步的探討。在共軛焦免疫螢光實驗中，觀察到DDX3在heat shock情況下，其形成的顆粒會與P body的標記蛋白DCP1a錯開，而會與stress granule的標記蛋白PABP共同座落於foci中。上述實驗證實DDX3是stress granule的組成蛋白之ㄧ。然而當DDX3 knockdown時，細胞中的stress granules仍可組裝，其完整性並不受影響，但其數量有下降的現象；同時，一般mRNAs及HuR的ligand c-fos mRNA仍可target 到stress granule。綜合以上的實驗結果，推測DDX3雖不是stress granule形成的必要因子，但仍有其重要的角色。因此，關於DDX3在stress granule中的生物功能，值得進一步的深入探討。

DDX3 belongs to the family of DEAD-box RNA helicase. DEAD box proteins play important roles in RNA processing ranging from transcription, pre-mRNA splicing, mRNA export to translation, and mRNA degradation. In our laboratory, studies of DDX3 have revealed that this protein can upregulate p21waf1/cip1 promoter. In addition, DDX3 was found to interact with pre-mRNA splicing factors. While present in the cytoplasm, DDX3 interacts with the translation initiation factor eIF4E that in turn inhibits the cap-dependent translation initiation. All these studies have indicated that DDX3 is a multifunctional RNA helicase. Recently, the study of Yedavalli et al. showed that DDX3 can directly interact with CRM1 (export receptor) or Rev (adaptor), and is involved in the HIV-1 Rev-CRM1 protein complex responsible for the nuclear export of unspliced and partially spliced viral transcripts. In view of these, this thesis focused on examining whether DDX3 plays a role in the export of cellular mRNAs.
In the CRM1-dependent mRNA export pathway, HuR serves as the adaptor protein which binds to the AU-rich mRNAs and escorts them to the cytoplasm. In this study, we investigated whether DDX3 involves in the HuR-CRM1-containing mRNP. The in vivo co-immunoprecipitation assay revealed that DDX3 interacts with HuR, and their interaction is greatly enhanced after heat shock treatment. Additionally, the confocal immunofluorescence experiment demonstrated that DDX3 and HuR colocalize to the specific cytoplasmic foci under heat shock condition. Furthermore, the possibility that DDX3 may associate with specific mRNA species, especially those AU-rich element (ARE)-containing transcripts were examined. By performing immunoprecipitation with Flag-tagged DDX3 and isolating the RNAs in the immunocomplex for the subsequent RT-PCR, it was found that under normal condition, DDX3 protein associates with its own mRNA, including the endogenous transcripts. However, after heat shock treatment, DDX3 was found to associate with some HuR ligands, such as c-fos, c-myc, and p21 mRNAs. All these mRNAs were bearing ARE in their 3’UTR. mRNA fluorescence in situ hybridization was also performed in DDX3 knockdown HeLa cells and the nuclear export of the CRM1-dependent mRNA c-fos was observed. However, the nuclear and cytoplasmic distribution for c-fos mRNA is not significantly affected upon knockdown of DDX3.
Furthermore, using confocal immunofluorescence analysis, we found that DDX3 proteins aggregate at some specific cytoplasmic foci in HeLa cells under heat shock condition. Interestingly, DDX3-formed foci are almost totally excluded from the P body marker DCP1a, but colocalizes with the stress granule marker PABP. This result suggested that DDX3 is one of the stress granule components. However, in DDX3 knockdown HeLa cells, the integrity of stress granule is not affected, although the number of stress granules appears decreasing. Moreover, both poly (A)+ mRNAs and HuR ligand c-fos mRNAs still target to stress granules. Therefore, it is suggested that DDX3, although not an essential factor, still plays an important role in stress granule. Therefore, to elucidate the biological role of DDX3 in stress granules, more extensive investigations are needed in the future.

Abstract (Chinese) 1
Abstract 3
Introduction 5
DEAD-box protein family of RNA helicase 5
DDX3 structure and biological functions 5
mRNA export pathway 6
CRM1-dependent mRNA export 8
General mRNA export 9
DEAD-box RNA helicases in mRNA export 10
Materials and Methods 13
Materials 13
Escherichia coli strain 13
Cell line 13
Plate and medium 13
Plasmids 14
Solutions 15
Chemicals 16
Enzymes 16
Primers and Oligonucleotide probes 16
Antibodies 17
Methods 18
Transformation 18
Plasmid purification 18
Cell culture and generation of DDX3 knockdown permanent cell line (mix population) 18
Transient transfection 18
Western blot 19
In vivo co-immunoprecipitation for Flag-DDX3 associated mRNPs and isolation of Flag-DDX3 associated mRNAs 19
RT-PCR 20
Confocal immunofluorescence 20
Fluorescence in situ hybridization (FISH) 21
Results 23
PartI Identify the adaptor protein associated with DDX3 23
PartII Identify the mRNA substrates associated with DDX3 25
PartIII Examine the role of DDX3 in mRNA export 27
PartIV Examine the cytoplasmic foci where DDX3 located
upon heat shock treatment 28
Discussion 32
The role of DDX3 in CRM1-dependent mRNA export 32
The relationship between DDX3 and the associated ddx3
mRNAs 35
The effects of heat shock on DDX3, HuR, and the associated mRNAs in stress granules 36
DDX3 is located in stress granule, but not P body 37
References 40
Tables and Figures 46

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