探討RNA解旋酶Fal1在90S前驅核糖體生合成中的功能及調控

Abstract

核糖體具有轉譯與蛋白質合成的關鍵功能，此複合體的生合成需依賴一系列有序的pre-rRNA裁切與大小次單元組裝。因此這個動態過程需眾多輔助因子協同參與，尤其DEAD-box RNA解旋酶能夠解開RNA二級結構，促進核糖核蛋白重塑，是核糖體生合成中不可或缺的角色。本研究探討ATP依賴性RNA解旋酶Fal1與兩個含MIF4G結構的調控因子Sgd1及eIF4G1，探討其於90S前驅核糖體組裝過程中的功能調控機制。我們發現抑制FAL1與SGD1表現，會導致細胞生長遲緩、pre-rRNA裁切異常及40S小次單元生成受阻。進一步透過Fal1 A67V (ATP 結合缺陷) 與D173N (解旋缺陷)突變，證實Fal1的ATPase與helicase活性，均為其功能必須。前人研究顯示Fal1、Sgd1與eIF4G1在體外可以形成穩定三聚體，三者亦具遺傳交互作用，缺失皆會影響pre-rRNA裁切與轉譯效率，且雙突變條件下更為嚴重。功能分析顯示，Sgd1能促進Fal1與RNA結合的能力，而eIF4G1則主要協助Fal1釋放RNA，促進完成解旋循環並從90S上離開。綜合研究結果，我們提出Fal1、Sgd1與eIF4G1可形成一個時序性的調控複合體，協助Fal1於90S核糖體中完成功能性循環，以維持40S小次單元生成與整體轉譯活性。

The ribosome has a key function in translation and protein synthesis in the cell. Its biogenesis relies on a series of orderly pre-rRNA cleavages and the assembly of both small and large subunits. This dynamic process requires the coordinated action of numerous accessory factors, among which DEAD-box RNA helicases play an essential role by unwinding RNA secondary structures and promoting the remodeling of ribonucleoproteins. In this study, we investigate the regulatory mechanism of the ATP-dependent RNA helicase Fal1 and two regulatory factors containing MIF4G domains, Sgd1 and eIF4G1, during the assembly of the 90S pre-ribosome. We found that repression of FAL1 or SGD1 expression led to growth retardation, abnormal pre-rRNA processing, and impaired formation of the 40S small subunit. Furthermore, mutations in Fal1, A67V (ATP-binding deficient) and D173N (helicase-deficient), also resulted in similar defects, confirming that both ATPase and helicase activities of Fal1 are essential for its function. Previous studies have shown that Fal1, Sgd1, and eIF4G1 can form a stable trimeric complex in vitro. These three proteins also exhibit genetic interactions, and their depletion affects pre-rRNA cleavage and translation efficiency. Moreover, the defects are more severe under double-mutant conditions. Functional analyses revealed that Sgd1 enhances Fal1’s RNA binding ability, whereas eIF4G1 primarily facilitates the release of RNA from Fal1, thereby promoting completion of the helicase cycle and release from the 90S pre-ribosome. Taken together, our results suggest that Fal1, Sgd1, and eIF4G1 form a temporally regulated complex that assists Fal1 in completing its functional cycle on the 90S ribosome, ensuring proper formation of the 40S small subunit downstream and maintenance of overall translational activity.