Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Splicing

Background:

• Saccharomyces cerevisiae Prp43 is a DEAH-box RNA-dependent ATPase.
• It catalyzes the release of excised lariat intron from the mRNA spliceosome.
• Previous studies showed mutations in motifs I, II, and VI are lethal and abolish ATP hydrolysis.

Purpose of the Study:

• To assess the effects of mutations in conserved residues in motifs Ia, IV, and V of Prp43.
• To identify key residues essential for Prp43 function in vivo and in vitro.
• To investigate the necessity and sufficiency of ATPase activity for Prp43 function.

Main Methods:

• Site-directed mutagenesis to create alanine and conservative substitutions in Prp43 motifs Ia, IV, and V.
• In vivo growth assays to assess lethality and dominant-negative phenotypes.
• In vitro assays to evaluate ATP hydrolysis and intron release inhibition.
• RNA/DNA duplex unwinding assays.

Main Results:

• Residues Arg150 (motif Ia), Phe309 (motif IV), Thr376, Leu383, and Thr384 (motif V) were identified as important for Prp43 function.
• Mutations in motif V (T376V, T384A, T384V) were lethal and dominant-negative in vivo, inhibiting lariat release in vitro.
• Mutant proteins T384A and T384V retained ATPase proficiency, indicating ATPase activity is necessary but not sufficient.
• Prp43 hydrolyzes all common NTPs and dNTPs, unwinds RNA/DNA duplexes in an ATP-dependent manner, requiring an RNA cofactor of >=20 nt.

Conclusions:

• Specific residues in Prp43 motifs Ia, IV, and V are critical for its biological function in mRNA splicing.
• While ATPase activity is essential, it is not sufficient for Prp43's role in lariat intron release.
• Prp43 possesses broader NTPase and RNA helicase activities, suggesting complex roles in spliceosome dynamics.