Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones.
The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Delta strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-DeltaP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.
Del Campo
Mark
M
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA.
Mohr
Sabine
S
Jiang
Yue
Y
Jia
Huijue
H
Jankowsky
Eckhard
E
Lambowitz
Alan M
AM
D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D007438:Introns; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D004272:DNA, Mitochondrial; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; C492152:DED1 protein, S cerevisiae; C058150:MSS116 protein, S cerevisiae; D053487:DEAD-box RNA Helicases