RNA helicase Drs1 gates 25S rRNA domain III incorporation during early nucleolar pre-60S maturation

Assembly of eukaryotic large ribosomal subunits (LSU) requires coordinated structural and compositional transitions within pre-60S particles, yet the underlying mechanisms remain poorly understood. Here, we show that the DEAD-box helicase Drs1 promotes early maturation across distinct regions of the pre-60S particle. Loss of Drs1 function causes accumulation of co-transcriptional intermediates retaining SSU processome components, indicating that Drs1 promotes timely separation of nascent LSU precursors from the small-subunit assembly pathway. Cryo-EM analyses reveal both a redistribution toward early nucleolar maturation states upon loss of Drs1, including Nsa1-deficient intermediates, and a confinement of Drs1-associated particles to states preceding stable incorporation of 25S rRNA domain III. Drs1 directly engages Erb1 through its unstructured N-terminal extension, promoting stable assembly of the Nop7-Erb1-Ytm1 module associated with domain III maturation. CRAC analysis localizes Drs1 to spatially clustered sites spanning the 5.8S and 25S rRNAs, encompassing domains I-IV. Together, these findings support a model in which Drs1 couples stabilization of assembly-factors with pre-rRNA remodeling across the pre-60S particle, thereby driving ordered early LSU maturation and the timed integration of domain III.