The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

In eukaryotes,the highly conserved u3 small nucleolar rna (snorna) base-pairs to multiple sites in the pre-ribosomal rna (pre-rrna) to promote early cleavage and folding events. binding of the u3 box a region to the pre-rrna is mutually exclusive with folding of the central pseudoknot (cpk),a universally conserved rrna structure of the small ribosomal subunit essential for protein synthesis. here,we report that the deah-box helicase dhr1 (ecm16) is responsible for displacing u3. an active site mutant of dhr1 blocked release of u3 from the pre-ribosome,thereby trapping a pre-40s particle. this particle had not yet achieved its mature structure because it contained u3,pre-rrna,and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the cpk. dhr1 was cross-linked in vivo to the pre-rrna and to u3 sequences flanking regions that base-pair to the pre-rrna including those that form the cpk. point mutations in the box a region of u3 suppressed a cold-sensitive mutation of dhr1,strongly indicating that u3 is an in vivo substrate of dhr1. to support the conclusions derived from in vivo analysis we showed that dhr1 unwinds u3-18s duplexes in vitro by using a mechanism reminiscent of dead box proteins. © 2015 sardana et al.