臺灣博碩士論文加值系統

Imporin-beta 蛋白質家族是細胞核質之間的運輸分子(nucleocytoplasmic transport receptors)，可攜帶特定的蛋白質或RNA穿越細胞核膜的孔洞。當細胞遭遇環境改變的刺激時，常啟動本身訊息傳遞的機制，改變基因的表達，以適應環境的變化。因此，我們首先想了解imporin-beta家族的成員，在面臨環境刺激時其分布是否改變，並藉此影響其他蛋白質在細胞中的分布。我們觀察到其中的transportin (TRN) 蛋白質受到環境變化的刺激，其分布會明顯的由細胞核改變到細胞質中的 stress granules。此外，少部分的TRN也會出現在processing bodies。stress granules 與processing bodies 皆為細胞質中的RNA聚集顆粒 (granules)：前者為細胞遭逢遽變時，無法進行轉譯的mRNA貯存之處；後者則聚集許多mRNA降解(decay)的酵素，可能為細胞質中mRNA降解之處。利用共軛焦顯微鏡作光漂白後螢光回復(Fluorescence Recovery After Photobleaching)實驗，我們發現TRN可在RNA聚集顆粒與細胞質間快速移動。使用RNA干擾(RNAi)的技術降低TRN表現，我們發現processing bodies的數目增加，stress granules的形成不受影響，此結果與細胞中缺乏mRNA 降解酵素 XRN1與Dcp1a時的情況相同。我們更進一步發現TRN可與 tristetraprolin (TTP)蛋白質及其調控之富含AU序列(ARE)的mRNA結合。TTP亦同時存在於processing bodies與stress granules中，當TTP結合至ARE mRNA，可促使RNA降解。以光漂白後的螢光回收率技術，我們發現TRN可能參與TTP在細胞質與RNA聚集顆粒間的運輸。當TRN表現量低時，ARE mRNA的半生期變得較長。因此，TRN可能參與TTP在細胞內的運送，進而影響RNA的降解。另一方面，我們也發現TRN可與細胞中微小RNA (microRNA) 及參與其作用之 Argonaute2(Ago2) 蛋白質結合；因此，TRN 亦可能參與細胞中微RNA 之調控。我們目前的證據，不僅顯示TRN可存在於細胞質中的RNA聚集顆粒，參與RNA結合蛋白質在期間之移動，並首度發現imporin-beta 家族成員參與ARE mRNA的調控亦可能參與細胞中微小RNA 之調控。

The distribution of proteins is likely to change in response to cellular stresses triggered signaling pathways. Since importin-beta family members are essential for nucleocytoplasmic transport of macromolecules, we attempted to explore whether importin-beta family proteins change their cellular localization in response to environmental change. Among the importin-beta members we analyzed, only transportin (TRN) was shown to detect in a subset of cytoplasmic processing bodies (P-bodies) under normal cell conditions and apparently translocate to stress granules (SGs) upon arsenite, heat shock or FCCP treatment of the cells. Both cytoplasmic granules contain translationally silenced mRNAs. SGs are site for accumulation of mRNA suffered stress-induced translational arrest and PBs are the sites for degradation of a subset of mRNAs and also for siRNA or miRNA-mediated gene expression silencing.
Fluorescence recovery after photobleaching analysis revealed that TRN moved rapidly in and out of cytoplasmic granules. Depletion of TRN enhanced P-body formation but did not affect the number or size of SGs, suggesting that TRN or its cargo(es) participates in cellular function of P-bodies. Accordingly, TRN associated with the AU-rich element (ARE)-binding protein, tristetraprolin (TTP), as well as its associated mRNAs. Depletion of TRN increased the number of P-bodies and stabilized ARE-containing mRNAs, as observed with knockdown of the 5’-3’ exonuclease Xrn1. Moreover, depletion of TRN retained TTP in P-bodies and meanwhile reduced the fraction of mobile TTP to SGs, indicating that TRN probably plays a role in trafficking of TTP between the cytoplasmic granules and whereby modulates the stability of ARE-containing mRNAs. Furthermore, we observed that TRN associated with microRNPs, which suggested a role of TRN in microRNA biogenesis or microRNA-mediated mRNA regulation. Taken together, our data indicated novel roles of TRN in cytoplasmic mRNA metabolism, primarily including mRNP trafficking to RNA granules and mRNA stability control.

Chapter 1 Introduction………………………………………………………………………………………………………………………1
1.1 Nucleocytoplasmic transport…………………………………………………………………………………………………1
General introduction of nucleocytoplasmic transport……………………………………………1
Cargo Recognition of nucleocytoplasmic transport receptors…………………………1
Regulation of nuclear transport receptors by cellular signaling……………2
Novel functions of importin-beta family……………………………………………………………………………3
1.2 Cytoplasmic RNA granules…………………………………………………………………………………………………………3
General introduction of cytoplasmic RNA granules……………………………………………………4
P-bodies, SGs and other RNA granules in yeast……………………………………………………………4
SGs and P-bodies in mammalian cells………………………………………………………………………………………6
Proteins involved in the formation of SGs or P-bodies………………………………………7
Current model of the formation of SGs and P-bodies………………………………………………7
1.3 The mRNA decay and silence pathways associated with P-bodies…………8
ARE-containing mRNA decay…………………………………………………………………………………………………………………9
microRNA mediated gene slicing……………………………………………………………………………………………10
1.4 Summary……………………………………………………………………………………………………………………………………………………11

Chapter 2 Materials and Methods……………………………………………………………………………………………12

Chapter 3 Results…………………………………………………………………………………………………………………………………21
3.1 TRN is located in both SGs and P-bodies………………………………………………………………21
TRN, importin-beta and importin 13 are located to cytoplasmic granules after ARS treatment……………………………………………………………………………………………21
TRN is located in P-bodies in normal cell condition and redistributed to SGs under cellular stresses…………………………………………………22
TRN moves rapidly in and out of the cytoplasmic granules……………………………23
Knockdown of TRN enhances the formation of P-bodies…………………………………………23
3.2 TRN interacts with TTP and participates in ARE-mRNA decay………………24
TRN associates and colocalizes with TTP………………………………………………………………24
TRN interacts with TTP associated mRNP and is involved in ARE RNA decay………………………………………………………………………………………………………………………………………………25
Depletion of TRN influences the distribution and movement of TTP in cytoplasmic granules…………………………………………………………………………………………………………26
3.3 TRN associates with microRNPs…………………………………………………………………………………………28
TRN is associated with Ago2 and miR-16…………………………………………………………………28
Depletion of TRN does not influence the maturation of miR-16………28
TRN associates with small RNAs………………………………………………………………………………………29
3.4 Identification of TRN-associated proteins…………………………………………………………29
Mass spectrum analysis of TRN associated proteins……………………………………29
The phosphorylation status of TRN and its associated proteins under stress conditions…………………………………………………………………………………………………………30
Chapter 4 Discussion……………………32
4.1 P-body and SG localization of TRN………………………………………………………………………………32
4.2 The roles of TRN in P-bodies……………………………………………………………………………………………33
4.3 The roles of TRN in regulation the traffic of TTP-associated mRNPs…………………………………………………………………………………………………………………………………………………………34
4.4 The roles of TRN in association with microRNA pathway…………………………36
4.5 Conclusion……………………………………………………………………………………………………………………………………………37

Reference…………………………………………………………………………………………………………………………………………………………38

Appendix……………………………………………………………………………………………………………………………………………………………48




List of Figures
Figure 1 Subcellular localization of importin-beta family members in
arsenite treated cells………………………………………………………………………………………………49
Figure 2 Subcellular localization of importin-beta family members
and SG-markers after arsenite treatment…………………………………………………50
Figure 3 Subcellular localization of TRN in arsenite, heat-shock or
FCCP treated cells…………………………………………………………………………………………………………51
Figure 4 Localization of TRN and SG-markers for stress granules after arsenite treatment…………………………………………………………………………………………………………………52
Figure 5 Localization of TRN in cytoplasmic P-bodies………………………………………53
Figure 6 Localization of TRN, importin b and P-body makers………………………54
Figure 7 Localization of importin b family members and P body marker
Dcp1a…………………………………………………………………………………………………………………………………………55
Figure 8 FRAP analysis of granule-localized proteins in arsenite-
treated cells………………………………………………………………………………………………………………………56
Figure 9 Comparison of the recovery kinetics of GFP-TRN, GFP-TIA1,
and GFP-mCPEB1 in cytoplasmic granules……………………………………………………57
Figure 10 Effect of TRN knockdown on the formation of SGs and P-
bodies……………………………………………………………………………………………………………………………………58
Figure 11 Effect of TRAP150, Lamin A/C, TRN and XRN1 knockdown on
the formation of P-bodies……………………………………………………………………………………59
Figure 12 The association of TRN and FLAG-TTP in HEK293 cells………………60
Figure 13 The interaction of GST-TRN and 35S-labbled TTP in GST-pull
down experiment………………………………………………………………………………………………………………61
Figure 14 Localization of TRN and FLAG-TTP in HeLa cells……………………………62
Figure 15 Effect of Ran on the interaction of TRN with TTP or HuR…63
Figure 16 Effect of Ran overexpression on the localization of FLAG-
TTP………………………………………………………………………………………………………………………………………………64
Figure 17 Subcellular distribution of TRN transport cargoes and FLAG-
TTP………………………………………………………………………………………………………………………………………………65
Figure 18 The association of TRN with ARE-containing RNA……………………………66
Figure 19 Association of FLAG-TTP to TRN and ARE containing-mRNAs……67
Figure 20 Effect of TRN knockdown on ARE-RNA decay……………………………………………68
Figure 21 Effect of TRN knockdown on the granule formation of FLAG-
TTP………………………………………………………………………………………………………………………………………………69
Figure 22 Effect of TRN knockdown on the granule formation of GFP-
TIA1……………………………………………………………………………………………………………………………………………70
Figure 23 FRAP analysis of GFP-TTP in TRN knockdown cells…………………………71
Figure 24 Partial co-migration and association of TRN with Ago2 and
miR-16………………………………………………………………………………………………………………………………………72
Figure 25 The association of TRN and small RNAs……………………………………………………73
Figure 26 Identification of TRN associated proteins…………………………………………74
Figure 27 The phosphorylation status of TRN and its associated
proteins under heat shock or arsenite treatment…………………………75



List of Tables
Table 1: Localization of importin-beta family members in P-bodies and stress granules (SGs) under normal and arsenite-stressed conditions………………………………………………………………………………………………………………………………………………76
Table 2 : Mass spectrometric identification of TRN associated proteins……………………………………………………………………………………………………………………………………………………………77

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