RNA chaperones stimulate formation and yield of the U3 snoRNA-Pre-rRNA duplexes needed for eukaryotic ribosome biogenesis.
Short duplexes between the U3 small nucleolar RNA and the precursor ribosomal RNA must form quickly and with high yield to satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells. These interactions, designated the U3-ETS (external transcribed spacer) and U3-18S duplexes, are essential to initiate the processing of small subunit ribosomal RNA. Previously, we showed that duplexes corresponding to those in Saccharomyces cerevisiae are only observed in vitro after addition of one of two proteins: Imp3p or Imp4p. Here, we used fluorescence-based and other in vitro assays to determine whether these proteins possess RNA chaperone activities and to assess whether these activities are sufficient to satisfy the duplex yield and rate requirements expected in vivo. Assembly of both proteins with the U3 small nucleolar RNA into a chaperone complex destabilizes a U3 stem structure, apparently to expose its 18S base-pairing site. As a result, the chaperone complex accelerates formation of the U3-18S duplex from an undetectable rate to one comparable with the intrinsic rate observed for hybridizing short duplexes. The chaperone complex also stabilizes the U3-ETS duplex by 2.7 kcal/mol. These chaperone activities provide high U3-ETS duplex yield and rapid U3-18S duplex formation over a broad concentration range to help ensure that the U3-precursor ribosomal RNA interactions limit neither ribosome biogenesis nor rapid cell growth. The thermodynamic and kinetic framework used is general and thus suitable for investigating the mechanism of action of other RNA chaperones.
Gérczei
Tímea
T
Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Shah
Binal N
BN
Manzo
Anthony J
AJ
Walter
Nils G
NG
Correll
Carl C
CC
D001681:Biological Assay; D005057:Eukaryotic Cells / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D020871:RNA Stability; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013816:Thermodynamics
C120533:IMP3 protein, S cerevisiae; C120534:IMP4 protein, S cerevisiae; D018832:Molecular Chaperones; D012322:RNA Precursors; D020537:RNA, Small Nucleolar; C117378:RNA, U3 small nucleolar; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins