- 學位論文
線蟲體內DEAD-box核糖核酸解旋酶F01F1.7 /DDX-23 在let-7生合成中所扮演的角色
The role of C. elegans DEAD-box RNA helicase F01F1.7/DDX-23 in let-7 microRNA biogenesis
摘要
DEAD-box RNA 解旋酶參與在許多改變RNA 結構或改變RNA 和蛋白交互作用的生化機制，其可能也包含了微型核醣核酸生合成以及發揮功能的某些步驟，但目前相關的研究並不多。在我們先前的RNA干擾篩選實驗中，發現藉由RNA 干擾將線蟲DEAD-box RNA 解旋酶F01F1.7/DDX-23的表現減低會加劇 let-7微型核醣核酸突變種let-7(mg279)的性狀，原因可能是減低F01F1.7的表現導致了成熟let-7微型核醣核酸的減少，在本篇論文中我們試圖探討F01F1.7是否可能在微型核醣核酸生合成中扮演著某種角色。我們發現減少F01F1.7/DDX-23表現量後也會減低其他微型核醣核酸的表現量，包括線蟲成熟過程中調控異時基因路徑 (heterochronic gene pathway)中的lin-4, miR-48, miR-241等。我們觀察到這些微型核醣核酸的初始物 (primary miRNAs) 在F01F1.7/DDX-23表現減少時有累積的現象，顯示這些初始微型核醣核酸似乎無法順利被Drosha 切割成前驅微型核醣核酸 (precursor miRNAs)。由於F01F1.7/DDX-23是酵母菌中訊息核醣核酸的PRP-28剪接因子 (pre-mRNA splicing factor) 的同源蛋白，減少F01F1.7/DDX-23表現時也有可能影響到初始微型核醣核酸處理過程中所需的 Drosha與Pasha的基因表現，然而我們卻沒有觀察到Drosha與Pasha的基因表現有任何降低的現象，顯示前述初始微型核醣核酸的累積並非因為Drosha與Pasha的表現量有所降低，而有可能是因為F01F1.7/DDX-23量降低後，Drosha無法順利切割處理這些初始微型核醣核酸所致。另外，除了與異時基因路徑相關的微型核醣核酸，我們也觀察到減少F01F1.7/DDX-23表現也導致一個與神經分化有關的微型核醣核酸lsy-6對其目標基因的調控有所降低。我們在線蟲中表現帶有GFP-Flag的F01F1.7/DDX-23 蛋白，並發現它如同預期地主要表現在細胞核。我們也試圖表現有抗原標記的Drosha蛋白，並利用免疫沈澱法來觀察是否F01F1.7/DDX-23跟含有Drosha的蛋白複合體有交互作用，但在本篇論文中我們並沒有成功表現該蛋白。我們在老鼠HEK293細胞中利用免疫沈澱也沒有觀察到Drosha與DDX23有交互作用。無論如何，我們的實驗結果顯示，F01F1.7/DDX-23可能幫助Drosha去切割處理某些初始微型核醣核酸，但其詳細機制仍須進一步的研究來闡明。
並列摘要
The DEAD-box RNA helicases are required for biological mechanisms with rearrangements of inter- or intra-molecular RNA structures and/or conformational changes of ribonucleoprotein complexes, which include microRNA biogenesis and function. However, little is known about the roles of DEAD-box RNA helicases in these miRNA processes. In our previous studies, we found that knockdown of a DEAD-box RNA helicase F01F1.7/DDX-23 in C. elegans enhanced let-7-related phenotypes in let-7(mg279) mutants, perhaps due to the reduced let-7 levels when F01F1.7/DDX-23 was depleted. Here, we seek further investigation in how F01F1.7/DDX-23 affects miRNA biogenesis. We found that depletion of F01F1.7/DDX-23 also reduced the levels of several miRNAs, in addition to let-7, involved in the C. elegans heterochronic gene pathway, including lin-4, miR-48 and miR-241. Depletion of F01F1.7/DDX-23 caused accumulation of the primary form of these miRNAs, suggesting reduced pri-miRNA processing by Drosha. Since F01F1.7/DDX-23 is an ortholog of yeast pre-mRNA splicing factor PRP-28, it is possible that depletion of F01F1.7/DDX-23 affected the expression of Drosha and its partner, Pasha. However, we found no decrease in their expression upon F01F1.7/DDX-23 knockdown. This suggests that depletion of F01F1.7/DDX-23 reduced the efficacy of pri-miRNA processing but not the levels of machinery. We also observed that depletion of F01F1.7/DDX-23 inhibited target regulation of the lsy-6 miRNA, which is involved in neuronal differentiation. We expressed GFP-Flag tagged F01F1.7/DDX-23 in C. elegans and found a predominately nuclear expression that was expected. We attempted to express tagged Drosha in C. elegans and examine possible protein interaction between Drosha and F01F1.7/DDX-23 but failed to establish stable strains. Instead, we examined this interaction in mouse HEK293 cells by co-immunoprecipitation and no evidence of interaction has been observed. Nevertheless, our results suggest that F01F1.7/DDX-23 facilitates Drosha function in the processing of some pri-miRNAs. The detailed mechanism will be a focus of our future studies.