臺灣博碩士論文加值系統

飲食限制(Dietary restriction, DR)在許多物種中皆被證實可以延長壽命並延緩老化。在先前以線蟲做為模式生物的研究中，發現sams-1基因在DR所造成壽命延長中扮演重要的腳色。sams-1所解碼出的蛋白質，能將ATP與甲硫胺酸結合產生甲基提供者SAM。在DR時，原本累積在腸道細胞核內的SAMS-1會出核，而SAMS-1在腸道細胞的核質比與壽命呈現負相關。為了探討sams-1是如何影響下游機制，進而造成壽命的延長，先前實驗室針對甲基轉移酶進行RNAi篩選，發現有部分RNA甲基轉移酶被抑制後，線蟲壽命延長。這些RNA甲基轉移酶的功能主要在幫助RNA進行加工、成熟。同時，在DR及抑制sams-1後，已知蛋白質合成會減少，且會使壽命延長，因此我們假設DR可能會減少RNA的成熟，造成蛋白質合成減少，最終使壽命延長。
而我主要針對mRNA的部分進行研究。在甲基轉移酶RNAi篩選中，找到使mRNA端帽甲基化的甲基轉移酶- tag-72。在先前的研究中已知tag-72的確在DR的下游。我利用帶有TAG-72鑲嵌紅螢光的線蟲株進行拍照，發現TAG-72主要表現在細胞核的核質中。接著利用dot blot實驗，我發現抑制tag-72後，的確可以使m7G-cap mRNA含量減少，由此確認tag-72的確有m7G-cap mRNA甲基轉移酶的功能。接著在SAMS-1減少及進行DR的情況下，也看到m7G-cap mRNA含量減少的現象。為了確認影響mRNA端帽甲基化後，是否也會影響mRNA後續加工。我們利用Fluorescence in situ hybridization (FISH)的方式觀察poly A mRNA在線蟲中的分布。結果發現在TAG-72減少、SAMS-1減少及DR的情況下，線蟲腸道細胞核核質的poly A mRNA都有減少。以上實驗證實了DR後，細胞核內的SAMS-1減少，減少了mRNA端帽的甲基化，也影響後續poly A加工，可能會造成被送出細胞核的成熟mRNA降低，使蛋白質生成下降，最終造成壽命延長。

Dietary restriction (DR) is the most robust intervention known to extend lifespan in a variety of species. Previous research showed that sams-1, which encodes the S-adenosyl methionine synthetase-1 (SAMS-1), mediates DR-induced longevity in Caenorhabditis elegans. SAMS-1 catalyzes the formation of a major cellular methyl group donor, S-adenosylmethionine (SAM), from methionine and ATP. The subcellular distribution of SAMS-1 depends on the levels of food consumption. The subcellular re-localization of SAMS-1 is required for DR-induced longevity. In order to identify the downstream effectors of SAMS-1 that may play roles in longevity regulation, we have previously performed a methyltransferase RNAi screening. From this screen, we have identified several candidate RNA methyltransferases, when knocked down, extends lifespan. Moreover, it has been shown that DR or sams-1 knockdown decreases protein synthesis in C. elegans and extends lifespan. To sum up, we propose that, in DR animals, decrease in SAMS-1 nuclear accumulation results in less SAM available for RNA methyltransferases, which in turn leads to reduced protein synthesis and extended lifespan.
My project focused on tag-72, a putative m7G-cap mRNA methyltransferase. Our previous studies suggest that tag-72 may be involved in DR-induced longevity. Thus, we aimed to examine the role of tag-72 in DR-induced longevity. We found that TAG-72 is mainly localized in the intestinal nucleoplasm. The level of m7G-cap mRNA decreased upon tag-72 RNAi, which confirms the function of TAG-72 as m7G-cap mRNA methyltransferase. Interestingly, there is also a reduced level of m7G-cap mRNA under DR or sams-1 inhibition. Next, we would like to know the effects of m7G-cap reduction on the mRNA processing. We found that the level of polyA mRNA in intestinal nucleoplasm is reduced upon tag-72 mutation, sams-1 RNAi or DR. Overall, we conclude that DR might attenuate SAM-dependent mRNA cap methylation, and subsequently reduce polyA processing. This may extend lifespan by lowering the level of mature mRNA and protein synthesis.

目錄
摘要 I
Abstract II
目錄 IV
圖目錄 VI
緒論 1
一、飲食限制(Dietary restriction, DR) 1
二、在飲食限制下關鍵基因 sams-1 3
三、RNA的成熟 4
四、生物體內蛋白質的恆定與壽命關係 7
研究目的 8
材料與方法 9
線蟲品系(strain) 9
線蟲培養(maintaining) 9
Escherichia coli OP50培養 10
Bleaching for eggs 10
AL / Starvation處理 11
RNA interference (RNAi)處理 11
線蟲壽命觀察 (lifespan) 12
線蟲中TAG-72分佈觀察 (TAG-72 distribution assay) 12
Fluorescence in situ hybridization (FISH) 13
Dot Blot 15
RNA isolation 17
Reverse Transcription (RT) 17
Quantitative Real-Time PCR 18
結果 19
1.觀察線蟲中TAG-72的分布及確認TAG-72的功能 20
2.飲食限制及SAMS-1的表現影響整體m7G mRNA含量 21
3.飲食限制及影響SAMS-1或TAG-72的表現改變腸道細胞核核質中poly A mRNA的含量 22
4.rRNA的成熟影響腸道細胞核核仁的大小 23
討論 25
圖表 28
參考文獻 48

圖目錄
Figure 1. RNAi screen下會延長壽命的甲基轉移酶……………………………….28
Figure 2. N2、EQ373、eat-2、EQ1123、EQ1124及EQ1125壽命觀察結果…...29
Figure 3. TAG-72在線蟲體內的分布………………………………………………30
Figure 4. N2處理tag-72 RNAi後對於整體m7G cap mRNA的影響…………….32
Figure 5. N2及VC1185整體m7G cap mRNA的差異…………………………….33
Figure 6. N2處理sams-1 RNAi後對於整體m7G cap mRNA的影響……………34
Figure 7. N2及EQ153整體m7G cap mRNA的差異……………………………...35
Figure 8. N2在正常飲食及飢餓2HR後整體m7G cap mRNA的差異…………...36
Figure 9. N2及AD1116整體m7G cap mRNA的差異…………………………….37
Figure 10. N2、VC1185、EQ153的poly A mRNA分布及定量結果…………….38
Figure 11. N2處理sams-1 RNAi後的poly A mRNA分布及定量結果…………..40
Figure 12. N2在正常飲食及飢餓2HR後的poly A mRNA分布及定量結果……41
Figure 13. N2及AD1116的腸道細胞核核仁大小定量結果……………………...42
Figure 14. N2及AD1116的腸道細胞核核仁大小定量結果………...……………43
Figure 15. N2、VC1185、EQ153的腸道細胞核核仁大小定量結果……………..44
Figure 16. N2處理nol-5、T07A9.8、trm-1 RNAi後腸道細胞核核仁大小定量結果……………………………………………………………………………………..45

表目錄
表一. QPCR primer表……………………………………………………………….46
表二. FISH分析及NP/C ratio統計結果總表……………………………………...46
表三. FISH分析及細胞核核仁大小統計結果總表………………………………..47

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