2019-01-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/665611摘要：核醣體生合成的量和細胞生長速度相關，細胞於對數成長期時，約70%的轉譯及轉錄皆用於生合成核醣體，以支持大量的蛋白質合成需求。很多核醣體蛋白具有核醣體以外的功能。過量或不足的核醣體蛋白表現皆會影響細胞生長。核醣體蛋白的生成必須根據rRNA的生成情形及環境刺激做緊密的調控。因此，分析核醣體蛋白生成的調控機制及其缺失時對核醣體生合成造成的影響，具有其重要性。在此研究中，將探討RNA結合蛋白如何調控Rpl43核醣體蛋白的生成。關於Puf6 及Loc1的細部研究，多集中在他們於ASH1 mRNA分布上的角色。他們被顯示和60S核醣體生合成亦相關，但機制未明。在我們之前的研究中發現，Puf6、Loc1和核醣體蛋白Rpl43形成三聚體，而且對維持Rpl43的蛋白質量及正確結合非常重要。關於失去Puf6及Loc1對Rpl43造成影響的機制，將會於本研究中做進一步探討。我們本來認為Puf6及Loc1會影響Rpl43的蛋白穩定性，但是發現這並非唯一的原因。Puf6及Loc1是RNA結合蛋白，能專一性的結合RPL43 mRNA。但是，他們並不影響RPL43 mRNA的量，因此，可能並非經由常見的Splicing機制進行調控。在目標1中，Puf6及Loc1於RPL43 mRNA的結合位及結合的可能調控機制將被分析。在目標2中，將分析Puf6及Loc1如何影響RPL43 mRNA的命運。在目標3中，將Puf6及Loc1從pre-60S核醣體上移除的因子將被定義，並進一步分析如果失去Rpl43、Puf6或Loc1時，將對核醣體生合成造成如何的影響。<br> Abstract: The level of ribosome biogenesis is highly connected with cell growth rate. In the log phase, in order to support abundant protein requirement, about 70% of transcription and translation are used for ribosome synthesis. Many ribosomal proteins have extra-ribosomal functions. Overexpression or insufficient supply of ribosomal proteins may impair the cellular growth. The supply of ribosomal proteins is tightly controlled in response to rRNA synthesis and environmental stimuli. Therefore, it is important to dissect the regulation mechanisms of the synthesis of ribosomal proteins and understand the defects caused by the mutation of this protein. The detailed studies about Puf6 and Loc1 focus on their roles in distribution of ASH1 mRNA. They have been also shown involved in 60S biogenesis but the mechanism is unclear. In our previous study, we found they form a ternary complex with ribosomal protein, Rpl43, and were critical to maintain the protein level and ensure the proper loading of Rpl43. The mechanisms about why absence of Puf6 or Loc1 caused under-accumulation and improper loading of Rpl43 will be dissected in this study. We first thought Puf6 and Loc1 may impact the protein stability of Rpl43, however, this is not the only reason. Puf6 and Loc1 are RNA binding proteins and can recognize RPL43 mRNA specifically. However, they did not change the level of RPL43 mRNA. Therefore, they may change the fate of RPL43 mRNA beyond splicing. In Aim 1, the binding motifs recognized by Puf6 and Loc1 and the signals regulate their associations with RPL43 mRNA will be examined. In Aim 2, how the fate of RPL43 mRNA changed by Puf6 and Loc1 will be analyzed. In Aim 3, the factor releases Puf6 and Loc1 from pre-60S will be identified and how the loss of Rpl43, Puf6 or Loc1 impacts the 60S biogenesis will be examined.核醣體生合成RNA結合蛋白輔助因子後轉錄調控轉譯調控ribosome biogenesisRNA binding proteinstransacting factorspost-transcriptional regulationtranslational control