Our study gives insight into the assembly pathway when nascent 40S and 60S subunits are still connected but begin to separate.

Synthesis of ribosomes begins in the nucleolus with 90S pre-ribosome formation, but already during this process the pre-40S and pre-60S pathways separate by pre-rRNA cleavages. However, how after this uncoupling the earliest pre-60S subunit continues to develop remains unknown. Here, we reveal a large subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyl-transferase Upa1-Upa2, large ?-solenoid scaffold modules, several RNA helicases amongst which Mak5 and two snoRNAs, C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing to visualize by electron microscopy a super 90S–pre-60S bipartite particle ~70 nm in size. Our study gives insight into the assembly pathway when nascent 40S and 60S subunits are still connected but begin to separate.