40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we developed an enhanced 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.终止密码子处的核糖体循环再利用，以支持后续多轮翻译过程，对高效蛋白质合成至关重要。60S亚基的移除由ATP酶（ATPase）Rli1（ABCE1）催化完成，而40S亚基的移除则被认为需要Tma64（eIF2D）、Tma20（MCT-1）以及Tma22（DENR）的参与。然而，目前仍不清楚这些Tma蛋白如何介导40S亚基的移除，并调控下游翻译的重新起始。本研究开发了一种增强型40S核糖体足迹分析策略，以直接观测细胞内核糖体回收的中间步骤。对编码这些Tma蛋白的基因进行敲除后，终止密码子处广泛积累未回收的40S亚基，直接证实了它们在40S亚基回收过程中的核心作用。进一步研究发现，Tma20/Tma22异二聚体介导了绝大多数40S亚基回收事件，而Tma64仅发挥次要功能。将一个自闭症相关突变引入TMA22后，40S亚基的回收活性丧失，这一结果将核糖体回收过程与神经系统疾病建立了直接关联。