RNA helicases mediate structural transitions and compositional changes in pre-ribosomal complexes

Abstract

Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.

Two molecules of Has1 RNA helicase function simultaneously in the biogenesis of small and large ribosomal subunits

“…The crosslinking pattern of the Has1-DAAD catalytic mutant showed strong reduction in 18S region, in agreement with its inability to bind 90S and pre-40S particles. Our data for the wild type Has1 are in a very good agreement with the recently published PAR-CRAC analysis (19). The observed crosslinking sites in the 5.8S/25S region also coincide well with the location of Has1 in several recent cryoEM structures of pre-60S ribosomes (27)(28)(29).…”

“…Interestingly, a new crosslinking site was detected in the 18S rRNA region, helices 4 and 6. This crosslinking pattern is in very good agreement with the recently published Has1 PAR-CRAC (19). Unfortunately, we were not able to crosslink the mutant Has1-DAAD using 4-TU for to us unknown reasons.…”

“…Using RNA-protein crosslinking, we identified multiple binding sites of Has1 on 18S, 5.8S and 25S rRNAs. Our data corroborate well the RNA binding sites reported in a new study published during preparation of this manuscript (19). Furthermore, we found that two copies of Has1 are temporarily present in 90S pre-ribosomes and remain associated with both pre-40S and pre-60S after the cleavage of pre-rRNA at the site A2.…”

“…The Has1-DAAD crosslinking produced overall ~3 times fewer reads compared to wild type. The crosslinking pattern remained similar to wild type at the 5' end of 25S rRNA (helices [17][18][19][20][21][22], but was strongly reduced in the 5.8S and 18S region. Several new peaks were present in the Has1-DAAD crosslinking profile ( Figure 3B and 3C).…”

Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

“…To date, 21 RNA helicases have been suggested to act during the process of ribosome biogenesis in yeast with eight implicated in SSU maturation, ten in LSU biogenesis and three in the assembly of both subunits [20]. A wealth of research has provided information on the binding sites and potential functions of many of these RNA helicases [21][22][23][24][25][26][27][28][29][30][31][32][33], with several RNA helicases implicated in promoting the release of specific snoRNPs from early pre-ribosomal particles [21,22,26,27,30,31] and others suggested to catalyse pre-rRNA rearrangements that induce formation of mature rRNA conformations facilitating the recruitment of RPs and/or AFs [23,32,34]. However, in the case of other RNA helicases that have been found associated with pre-ribosomal particles, little is known about the timing of their association, the interactions they make within pre-ribosomes and what contribution their activity makes to the assembly process.…”