The project’s first task aimed at the structural basis of the assembly of the human 40S small ribosomal subunit by single particle cryo-EM. Here, we could provide information of the last cytoplasmic steps of human pre-40S assembly (Ameismeier et al., Nature, 2020) which includes the decisive ultimate cleavage of the rRNA to its mature 18S state by a dedicated nuclease. In addition, we succeeded in visualizing the complete nucleoplasmic phase of 40S subunit maturation (see Figure, Cheng et al., NAR, 2022) which is mainly characterized by the formation of the so-called head domain of this subunit. Since we visualized a large collection of human and of yeast intermediates, we were in the position to compare and noticed to our surprise that there are actually rather small differences between the assembly pathways of the species during this phase which might be a general evolutionary feature.
The next task aims at the structural elucidation of human 60S large ribosomal subunit assembly. We succeeded to isolate another set of nuclear intermediates on the basis of a mutation in the assembly factor NLE1 (Rsa4 in yeast). These intermediates represented rather late nuclear particles, which showed again that there is large a degree of conservation when compared to the known yeast intermediates (manuscript in poreparation). Another aspect of 60S maturation, the incorporation of the so-called 5S RNP and its signalling to the P53 stress surveillance system has been successfully analysed and provided first insights into how the 5S RNP is prepared for 60S incorporation and, in case of accumulation, sequesters MDM2 in order to stabilize P53 (Estrada et al., Nat. Struct. Mol. Biol., 2023). The unexpectedly high degree of conservation between human and yeast maturation justified to address special aspects of 60S subunit maturation again in the easy-to-handle fungal model with the high probability to gain insights which both systems have in common. We could thereby visualize the so far earliest 5S RNP incorporation in the fungal system (Lau et al., EMBO Rep., 2023) and te remodelling of the pre-60S subunit by the ATPases Rea1 Spb4 (Mitterer et al., eLife, 2023).
