title: The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.
abstract: 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.
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Richa
authors.initials: R
authors.lastname: Sardana
authors.affiliation: Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine & Science, North Chicago, Illinois, United States of America.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: Sander
authors.initials: S
authors.lastname: Granneman
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Jieyi
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Gill
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Ophelia
authors.initials: O
authors.lastname: Papoulas
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Marcotte
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Tollervey
authors.affiliation: Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine & Science, North Chicago, Illinois, United States of America.
authors.forename: Carl C
authors.initials: CC
authors.lastname: Correll
authors.affiliation: Department of Molecular Biosciences and the Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Arlen W
authors.initials: AW
authors.lastname: Johnson
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D003080:Cold Temperature; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012322:RNA Precursors; D012337:RNA, Ribosomal, 18S; D020537:RNA, Small Nucleolar; C117378:RNA, U3 small nucleolar; D029701:Saccharomyces cerevisiae Proteins; C414169:ECM16 protein, S cerevisiae; D053487:DEAD-box RNA Helicases