裂殖酵母Rpl27旁系同源蛋白特异性介导饥饿状态下核糖体大亚基60S核仁存储的机制和功能探究

The abundance of ribosome, the highest energy consumption cellular organelle, is carefully adjusted in response to changes in nutrient availability. Two strategies have been identified for cells to down regulate its ribosome abundance during starvation: reducing ribosome biogenesis and degrading ribosomes by a selective autophagic pathway. . Our preliminary investigation in fission yeast has found that upon starvation, ribosome large subunit proteins (Rpls) are enriched in nucleolus whereas small subunit proteins are enriched in cytoplasm, suggesting that spatial separation of 60S and 40S ribosomal subunits may also serve the purpose of adjusting the total protein translation capacity. Noticeably, in stationary phase cells, Rpl2701 is exclusively nucleolus-localized whereas its functional paralog Rpl2702 is exclusively in cytoplasm, exhibiting distinct paralog-specificity. . Thus, these preliminary data support a working model that selective blockage of pre-60S large subunit in nucleolus as storage mediated by paralogous ribosome protein genes (rpl27 paralogs at least) is a novel strategy in fission yeast to adjust ribosome abundance during starvation. . We plan to test and verify this hypothesis extensively using genetic, biochemical and cell biology approaches. We will investigate the signal pathways that control the nucleolus storage of Rpl2701-containing pre-60S. We will also investigate the protein structural feature unique for Rpl2701 in distinction to Rpl2702 that allows it responds to the upstream signal specifically. We will further explore the physiological significance of this novel pathway to adjust protein translation capacity and its relationship with the two known pathways - ribosome biogenesis and ribosome degradation.. The success of this proposal will establish a novel strategy for cell to adjust its ribosome abundance during starvation and provide the evidence for the first time that heterogeneous ribosomes are functionally different in response to environmental cues.

在细胞响应外界营养条件变化过程中, 核糖体作为一个高能耗细胞器，其丰度是被精确调控的。目前已知细胞在饥饿状态下，下调自身核糖体丰度主要有两种方式：抑制新核糖体的合成和促进现有核糖体的降解。我们初步的研究结果提示，裂殖酵母细胞在饥饿状态下可能存在第三种途径调节自身核糖体丰度: 即将部分核糖体大亚基 60S亚基存储在核仁中，实现60S与40S亚基在空间上的隔离，从而下调 80S核糖体丰度。. 本项目将利用生物化学和细胞生物学手段研究异质性核糖体60S大亚基核仁存储现象和存在形式，探究核仁存储的发生机制及异质性核糖体介导核仁存储的分子机制。本项目的成功将揭示细胞饥饿状态下调控核糖体丰度的一种新途径，并且阐明通过核糖体异质性调节核糖体丰度的分子机制。

在细胞响应外界营养条件的变化过程中，核糖体作为能量消耗最高的细胞器，其丰度是被多种策略精确调控的，包括新核糖体合成的抑制、现有核糖体的降解和核糖体冬眠。我们在裂殖酵母中的研究，发现了一种新的、通过核糖体大小亚基的空间分离调控核糖体丰度的途径。我们发现细胞从介于对数生长期和平台期之间的过渡时期（transition phase）开始下调细胞质中核糖体的丰度。具体就是，由Rpl2701介导大部分pre-60S大亚基存储在核仁，而Rpl2702介导的少量pre-60S运输到细胞质，与那里的40S小亚基组装成成熟的80S核糖体，维持细胞的基本功能。这种选择性的pre-60S大亚基核仁存储可能是由pre-60S成熟和核质转运相关的核糖体组装因子Tif6调控的。因为Tif6能特异性地与Rpl2701介导的pre-60S结合。我们发现，过表达Tif6蛋白，当细胞进入平台期时，能扰乱pre-60S大亚基在核仁存储的细胞比例。我们进一步发现，当外界营养条件恢复后，核仁中储存的pre-60S大亚基可被迅速释放到细胞质中。因此，我们认为pre-60S大亚基在核仁的部分保留代表了一种快速调节蛋白质合成能力的反应机制，与核糖体冬眠途径和其他需要长时间反应的核糖体降解和新核糖体合成抑制信号通路一起，共同调节核糖体丰度。