探討RACK-1在線蟲終端細胞分化及let-7 miRNA表現中扮演的角色

The role of receptor for activate C kinase RACK-1 in terminal cell differentiation and let-7 miRNA level in Caenorhabditis elegans

王維杰(Wei-Chieh Wang)

指導教授 ： 詹世鵬

國立臺灣大學/醫學院/微生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.01476

摘要

線蟲的發育受到異時基因調控路徑 (the heterochronic gene pathway) 中一些重要因子所調控，包括數個微型核醣核酸 (microRNA)，其中 let-7 微型核醣核酸在線蟲從第四幼蟲期到成蟲的過渡轉變中扮演重要角色。let-7 微型核醣核酸調控細胞週期脫離與終端分化，低量的 let-7 表現會造成線蟲外陰發育異常及上皮接縫細胞 (hypodermal seam cell) 分化遲緩，導致成蟲外陰爆裂以及上皮接縫細胞持續分裂的性狀，而在人類中低量的 let-7 表現則與許多類型的癌症發生有關。先前研究認為 receptor of activated C Kinase 1 (RACK-1) 這個鷹架蛋白 (scaffold protein) 會幫助微型核醣核酸沈默複合體 (miRISC) 與核糖體結合，促進線蟲及人類的 let-7 微型核醣核酸發揮其功能。然而，在本篇論文中我們卻發現以核醣核酸干擾 (RNA interference) 降低線蟲體內 RACK-1 表現時，具有亞等位基因 (hypomorphic gene) let-7(n2853) 突變株所表現的外陰爆裂以及上皮接縫細胞持續分裂的性狀被抑制，相同的突變性狀抑制現象也出現在同時缺乏兩個 let-7 旁系同源微型核醣核酸 miR-48 及 miR-241 的突變株之中，顯示減低 RACK-1 基因表現反而可能促進與 let-7 相關的基因調控。另外，作為 let-7 數個重要目標基因之一的 hbl-1 發生失能突變時可導致某個程度的早熟性狀，而減低 RACK-1 基因表現時此 hbl-1(mg285) 突變株中早熟性狀則會更進一步被加強，該現象也許可以被解釋成 hbl-1(mg285) 突變本身與加強 let-7 抑制其他目標基因的加成效應。我們也發現，減低 RACK-1 基因表現對 let-7 突變性狀的抑制需要微型核醣核酸沈默複合體中的核心蛋白 Argonaute ALG-1，顯示該效應的確是經過微型核醣核酸的功能而發揮作用的。我們進一步發現，減低 RACK-1 基因表現可以導致成熟 let-7 量上升，這或許可以解釋我們所看到的突變性狀抑制現象。在 RACK-1 及 Dicer 雙重核醣核酸干擾實驗中，減低 Dicer 表現造成前驅 pre-let-7 微型核醣核酸無法正常被切割成成熟 let-7 而累積，此時再減低 RACK-1 表現可看到更多的 pre-let-7 累積現象，顯示 RACK-1 可能影響了一個在 Dicer 上游的 let-7 微型核醣核酸生合成步驟。我們探討幾個與 pri-miRNA 轉錄後加工有關的蛋白 ADAR、Tudor-SN及KSRP 是否可能參與在 RACK-1 調控 let-7 miRNA生合成的機制，發現在線蟲 ADR-2 突變株中減低 RACK-1 表現不會導致 pre-let-7 量的增加，可能暗示 ADR-2 與 RACK-1 影響 let-7 微型核醣核酸生合成機制有所關連，但其機制仍然留待未來的研究去闡明。

關鍵字

線蟲 ； let-7 ； 微小核糖核酸 ； RACK1 ； heterochronic

並列摘要

The C. elegans heterochronic gene pathway orchestrated by regulation of several miRNAs and their targets regulates larval stage-specific development. The let-7 miRNA controls cell cycle exit and terminal differentiation at the larva-to-adult transition. Low expression of let-7 results in abnormal vulva development and attenuated terminal differentiation of hypodermal seam cells in C. elegans, causing vulva bursting and reiterated seam cell division in adult worms, and also has been associated with a variety of cancers in humans. Previously, the scaffolding receptor of activated C kinase (RACK-1) has been proposed to facilitate the recruitment of miRNA-induced silencing complex (miRISC) to the polysome and the let-7 function in C. elegans and humans. However, here we found that RACK-1 knockdown by RNAi suppressed the abnormal vulva and seam cell phenotypes caused by a hypomorphic let-7(n2853) allele or lack of the let-7 paralogs miR-48 and miR-241, suggesting that, opposing to the previous studies, depletion of RACK-1 may enhance let-7 function. Moreover, the incomplete precocious phenotypes caused by hbl-1(mg285), the null allele of one of several important let-7 targets, was enhanced by RACK-1 knockdown. This might be explained by a synthetic effect of the hbl-1(mg285) mutation and enhanced let-7 down-regulation on other targets. In addition, we found that the suppression on let-7 mutants by RACK-1 knockdown is dependent on the Argonaute protein ALG-1, the core of miRISC, suggesting that RACK-1 functions via the miRNA regulation machinery. We further demonstrated that depletion of RACK-1 caused increased levels of mature let-7 that may explain the mutant suppression effect. In RACK-1/Dicer double RNAi experiments, we observed that depletion of RACK-1 reinforced the accumulation of pre-let-7 caused by Dicer knockdown, suggesting that RACK-1 affects a let-7 biogenesis step upstream of the Dicer processing. We examined the role of ADAR, Tudor-SN and KSRP proteins that have been implicated with post-transcriptional modification of pre-miRNAs in the putative RACK-1 function regarding miRNA biogenesis and found that depletion of RACK-1 in an ADR-2 null mutant caused no increase in the pre-let-7 levels. This might lead to the involvement of ADR-2 with RACK-1 function in miRNA biogenesis. The detailed mechanism awaits future elucidation.

並列關鍵字

C. elegans ； let-7 ； miRNA ； RACK1 ； heterochronic

參考文獻

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