Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22.
The DEAH protein Prp22 is important for the second transesterification step of pre-mRNA splicing, and it is essential for releasing mature mRNA from the spliceosome. Recombinant Prp22 has RNA-stimulated ATPase and ATP-dependent unwinding activities, which are crucial for the mRNA release step. In this study, we characterize the RNA-binding, NTP hydrolysis, and RNA unwinding functions of Prp22. Using nitrocellulose filter binding assays, we determined that the apparent affinity of Prp22 is approximately 20-fold greater for single-stranded RNA than for single-stranded DNA or duplex nucleic acids. Inclusion of hydrolyzable ATP in binding reactions increased the apparent K(D) for RNA by 3-4-fold. The Prp22-RNA interaction is influenced by the length of the RNA chain, and the apparent K(D) values for poly(A)(40) and poly(A)(10) are 17 and 140 nM, respectively. RNA-stimulated ATP hydrolysis is similarly affected by chain length, and optimal activity requires RNA oligomers of >or=20 nt. We show that Prp22 can hydrolyze all common NTPs and dNTPs with comparable efficiencies and that Prp22 unwinds RNA duplexes with 3' to 5' directionality.
Tanaka
Naoko
N
Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.
Schwer
Beate
B
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000737:chemistry; D053487:DEAD-box RNA Helicases; D004265:DNA Helicases / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D043583:Nucleoside-Triphosphatase / Q000737:chemistry*; D011061:Poly A / Q000737:chemistry; D020365:RNA Helicases / Q000737:chemistry*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017381:Spliceosomes / Q000201:enzymology*; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000737:chemistry
D009692:Nucleic Acid Heteroduplexes; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D011061:Poly A; C015465:poly(dA); D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C067135:PRP22 protein, S cerevisiae; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D014544:Uridine Triphosphate