探討 DjCBC-1 蛋白於渦蟲神經系統與成體幹細胞恆定性之調控功能

Abstract

生物藉由再生的過程以長回失去的組織，或更新體內老化細胞來維持個體原 本正常功能。扁形動物中的渦蟲具有強大的再生能力，利用體內廣布的多功能成體 幹細胞(neoblasts)，在受傷時增生並分化成所有類型的細胞來修復失去的組織。 許多基因參與成體幹細胞的增生與分化，這些基因都需要在轉錄或後轉錄層級精 確地被調控，以維持成體幹細胞正常功能。在 DEAD-box RNA 解旋酶家族的成員 中，DDX6 的同源蛋白於不同真核生物中都具有高度保守性。過去的研究指出， DDX6 透過降解 mRNA 和抑制轉譯的方式調控哺乳類上皮幹細胞及神經幹細胞。 先前研究顯示，渦蟲的 DDX6 同源基因 DjCBC-1 高度表現在成體幹細胞及神經系 統，以 RNAi 方式減弱 DjCBC-1 的表現會降低渦蟲再生時的存活率、改變渦蟲腦 部發育與功能。本研究論文以高度專一性的抗 DjCBC-1 抗體驗證 DjCBC-1 蛋白的 渦蟲全身性分佈，尤其高度表現於腦及再生組織中，與先前以原位雜合偵測 mRNA 的結果相似。此外 DjCBC-1 在細胞內分布於細胞核與細胞質聚集點。利用此抗體 進行免疫沈澱，成功純化分離 DjCBC-1 的 RNA-蛋白複合體，分析其中的 RNA 組 成，顯示 DjCBC-1 結合並調控專一表現於 neoblast 的 mRNA，藉此影響成體幹細 胞的數量與分化。

Regeneration is a process of regrowing the missing body parts or renewing aging cells to preserve the normal functions of an organism. Regeneration in planarians requires the somatic stem cells, named neoblasts, which proliferate and differentiate into all types of cells. Many genes involved in regeneration are regulated transcriptionally or post- transcriptionally. DEAD-box proteins are one of the post-transcriptional regulators. DDX6 is a highly conserved DEAD-box protein in eukaryotes. Studies showed that DDX6 regulates differentiation of epidermal and neural stem cells in mammals, by regulating mRNA degradation and/or translational repression. In planarian, the DDX6 ortholog, DjCBC-1, is highly expressed in neoblasts and neurons. Depletion of DjCBC- 1 decreased the survival rate, perturbed the brain structure, and delayed the formation of eyespots during regeneration. Long-term depletion of DjCBC-1 by RNAi also abolished the negative phototaxis of planarian. Here, I examined the expression of DjCBC-1 at the protein level. DjCBC-1 protein is expressed ubiquitously and is highly expressed in the brain and regenerating tissue. This finding is consistent with previous results, which were observed using whole-mount in situ hybridization (WISH). DjCBC-1 is localized to the nucleus and cytoplasm foci at the subcellular level. The depletion of DjCBC-1 increased the number of neoblasts. Finally, using an antibody specifically recognizing DjCBC-1, I immuno-precipitated DjCBC-1 RNA-protein complex and pulled down the neoblast- specific RNAs. My results suggested the function of DjCBC-1 in regulating target mRNA levels and participating in the maintenance of neoblasts.