Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro.
The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended alpha-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted alpha-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the alpha-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.
Mohr
Georg
G
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712, USA.
Del Campo
Mark
M
Mohr
Sabine
S
Yang
Quansheng
Q
Jia
Huijue
H
Jankowsky
Eckhard
E
Lambowitz
Alan M
AM
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000737:chemistry; D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D020868:Gene Silencing; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D066298:In Vitro Techniques; D007438:Introns; D007526:Isoelectric Point; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010957:Plasmids; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D024361:Amino Acids, Basic; D029721:Drosophila Proteins; D018832:Molecular Chaperones; D016337:RNA, Catalytic; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D000251:Adenosine Triphosphatases; C492152:DED1 protein, S cerevisiae; C058150:MSS116 protein, S cerevisiae; C088269:vas protein, Drosophila; D053487:DEAD-box RNA Helicases