Musalgaonkar Sharmishtha, Yelland James N, Chitale Ruta, Rao Shilpa, Ozadam Hakan, Taylor David W, Cenik Can, Johnson Arlen W
Department of Molecular Biosciences, The University of Texas at Austin, Austin, USA.
SalioGen Therapeutics, Lexington, USA.
Nat Commun. 2025 Feb 3;16(1):1278. doi: 10.1038/s41467-025-55844-8.
Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel of the pre-60S subunit. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation. Using cryo-electron microscopy to image Reh1-bound 80S ribosomes, we found they contain A site peptidyl tRNA, P site tRNA and eIF5A, indicating that Reh1 does not dissociate from 60S until translation elongation. We propose that Reh1 is displaced by the elongating peptide chain, making it the last assembly factor released from the nascent 60S subunit during its initial round of translation.
功能性核糖体亚基的组装并将其成功递送至翻译池是蛋白质合成和细胞生长的先决条件。在酿酒酵母中，核糖体组装因子Reh1在60S前体亚基细胞质成熟的后期与其结合。先前的研究表明，Reh1的C末端插入60S前体亚基的多肽出口通道。在此，我们表明与Reh1结合的新生60S亚基与40S亚基结合形成活跃翻译的核糖体。使用选择性核糖体谱分析，我们发现与Reh1结合的核糖体聚集在起始密码子附近的开放阅读框中。与Reh1结合的核糖体中起始tRNA也大量富集，表明它们与早期延伸相关。使用冷冻电子显微镜对与Reh1结合的80S核糖体进行成像，我们发现它们含有A位点肽基tRNA、P位点tRNA和eIF5A，这表明Reh1直到翻译延伸阶段才从60S上解离。我们提出Reh1被延伸的肽链取代，使其成为新生60S亚基在第一轮翻译过程中最后释放的组装因子。
