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Updated: Apr 22, 2026
ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast
Published on: June 30, 2022
Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing
Agata Jacewicz1, Beate Schwer2, Paul Smith1
1Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Abstract:
Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1-89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127-588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127-588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.
Insights
Yeast Prp28, a splicing factor, has a catalytic domain with RNA-dependent ATPase activity. Key residues in ATP and RNA binding sites are essential for its function in pre-mRNA splicing.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Yeast Prp28 is a DEAD-box protein crucial for pre-mRNA splicing.
- It functions by displacing U1 small nuclear ribonucleoprotein (snRNP) from the 5' splice site.
Purpose of the Study:
- To characterize the structure and function of Yeast Prp28.
- To identify key residues and domains essential for its RNA-dependent ATPase activity and in vivo function.
Main Methods:
- Protein expression and purification of recombinant Prp28 and its C-terminal domain.
- Crystallography to determine the structure of Prp28-(127-588) bound to AMPPNP•Mg2+.
- Alanine scanning mutagenesis to assess the in vivo importance of specific amino acid residues.
- Analysis of dominant-negative growth defects caused by mutant Prp28 overexpression.
Main Results:
- Yeast Prp28 consists of a N-terminal segment and a C-terminal catalytic domain with RNA-dependent ATPase activity.
- The crystal structure of the catalytic domain revealed a conformation poised for ATP binding but not hydrolysis.
- Specific residues in the ATP-binding (e.g., Asp341, Glu342) and RNA-binding sites (e.g., Arg476) are critical for Prp28 activity.
- Synthetic lethality revealed functional redundancy in ATP- and RNA-binding sites.
Conclusions:
- The structure and mutagenesis data elucidate the mechanism of Yeast Prp28 in pre-mRNA splicing.
- Essential residues and functional redundancies within the ATP and RNA binding sites have been identified.
- Defective ATP-site mutants exhibit dominant-negative effects, impacting cell growth.
