線蟲體內蛋白LIN-28結合蛋白鑑定

Abstract

LIN28為核醣核酸結合蛋白，最早在線蟲被發現，調控線蟲的發育。在高等生物中，LIN-28在發育、多能性(pluripotency)、腫瘤生成、代謝...等都扮演重要角色。LIN-28在幹細胞/前驅細胞中高度表現，抑制let-7 微小核醣核酸表現，消除let-7對其下游目標的抑制(如細胞週期因子)，促使細胞增生。LIN-28在哺乳類的同源蛋白為LIN-28A 和 LIN-28B，分別在細胞核與細胞質中與pri-let-7 微小核醣核酸或pre-let-7 微小核醣核酸結合，阻止其生合成，最終影響let-7 微小核醣核酸的量。但是關於LIN-28是如何作用、有甚麼共同因子一起促進其作用或抑制其作用，至今仍有許多地方尚未釐清。 在我的論文中主要是想要在線蟲(C. elegans)體內探討LIN-28是否有其他蛋白質會與LIN-28結合，再進一步探討它們是如何參與在LIN-28調控基因表現的過程。我的論文策略主要是想利用免疫沉澱法來鑑定LIN-28的結合蛋白。原理是利用抗HA單株抗體與線蟲萃取液中的LIN-28::GFP-flag-2HA 進行免疫沉澱，將LIN-28分離出來並利用一維以及二維蛋白質電泳分析及基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF/TOF MS) 和液相層析串聯式質譜儀(LC MS/MS)分析得到可能與LIN-28有交互作用的蛋白質成分。 針對所有被免疫沉澱法純化出來的候選蛋白質，我們利用核醣核酸干擾(RNA interference)降低其基因表現並觀察其產生的性狀。我們發現其中一個候選蛋白的基因hrp-2在核醣核酸干擾基因沉默之後會出現外陰(vulva)發育缺失、接縫細胞(seam cell)分化異常和翼(alae)斷裂等類似let-7突變性狀的現象，但抑制hrp-2的表現對於let-7的生合成並沒有影響，是否HRP-2參與在LIN-28 或let-7 調控路徑執行功能則是還要再進一步去證實。

LIN-28, a RNA-binding protein, has emerged as a modulator of the processing of the let-7 microRNA. This role for LIN-28 has important implication for our mechanistic understanding of pluripotency, the timing of development, and oncogenesis. LIN-28 was first characterized in the Caenorhabditis elegans as an important regulator of developmental timing. The mammalian homologs of LIN-28, LIN-28A and LIN-28B, bind to the terminal loop of the precursors of let-7 family miRNAs and block their processing into mature miRNAs. Here we aim to identify specific factors that associate with LIN-28 in C. elegans. We use anti-HA monoclonal antibody immunoprecipitation to isolate LIN-28 along with its associating proteins and then analyze these proteins by 1-D or 2-D electrophoresis. Further we use RNAi to reduce the gene expression of the candidate LIN-28-binding proteins and investigate their genetic interaction with LIN-28 and/or let-7. Our results indicate that knockdown of hrp-2, which encodes a candidate LIN-28- binding protein found in our immunoprecipitation assay, resulted in let-7 defective phenotypes, such like seam cell differentiation defects, retarded alae formation and vulva protruding. However, the level of let-7 was not altered when knocking down hrp-2. The role of HRP-2 in the LIN-28 or let-7 regulation pathway is still unclear and awaited to be further investigated.