Eukaryotic ribosome assembly requires the coordinated processing and extensive remodeling of pre-rRNAs. During late nuclear maturation of the 60S subunit, sequential removal of the internal transcribed spacer 2 (ITS2) is initiated by endonucleolytic cleavage at site C2 by the conserved Las1 nuclease. Las1 acts together with the kinase Grc3 and the Rix1 complex to form the Rixosome, which also functions in transcriptional regulation. However, the assembly of the Rixosome, its recruitment to pre-ribosomes, and its activation for ITS2 cleavage remain unclear. Here, we present cryo-EM structures of the human LAS1 complex, two structures of the isolated Rixosome and nine transition states of Rix1-bound pre-60S particles from Schizosaccharomyces pombe. These structures reveal a dynamic Rixosome architecture in which the heterotetrameric Las1 complex engages one or two copies of the Rix1 complex. Rix1 binding is highly flexible in the human Rixosome but rigid in the yeast complex. The isolated yeast Rixosome remains inactive, but binding to the pre-60S particle triggers a structural rearrangement that allows for substrate engagement and activation of the nuclease. Together, our results define the dynamic architecture of the Rixosome and provide a structural framework for ITS2 processing during nuclear maturation of the eukaryotic 60S ribosomal subunit.
M. Thoms, Jing-Yi Zhu, Xia-Ying Hong et al.· bioRxiv· 0 citations
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.
M. Thoms, Sanem Ayaz-Kök, Kohei Abe et al.· bioRxiv· 0 citations
mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3’-5’ helicase activity and pulling on the mRNA from its 3’ end.
L. Chitoiu, T. Denk, Martin B. D. Müller et al.· bioRxiv· 0 citations
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