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Structure and function of the PWI motif: a novel nucleic acid-binding domain that facilitates pre-mRNA processing
Blair R Szymczyna1, John Bowman, Susan McCracken
1Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada M5G 2M9.
Abstract:
The PWI motif is a highly conserved domain of unknown function in the SRm160 splicing and 3'-end cleavage-stimulatory factor, as well as in several other known or putative pre-mRNA processing components. We show here that the PWI motif is a new type of RNA/DNA-binding domain that has an equal preference for single- and double-stranded nucleic acids. Deletion of the motif prevents SRm160 from binding RNA and stimulating 3'-end cleavage, and its substitution with a heterologous RNA-binding domain restores these functions. The NMR solution structure of the SRm160-PWI motif reveals a novel, four-helix bundle and represents the first example of an alpha-helical fold that can bind single-stranded (ss)RNA. Structure-guided mutagenesis indicates that the same surface is involved in RNA and DNA binding and requires the cooperative action of a highly conserved, adjacent basic region. Thus, the PWI motif is a novel type of nucleic acid-binding domain that likely has multiple important functions in pre-mRNA processing, including SRm160-dependent stimulation of 3'-end formation.
Insights
The PWI motif is identified as a novel nucleic acid-binding domain, crucial for SRm160 protein function in pre-mRNA processing. This discovery sheds light on its role in RNA binding and 3'-end formation.
Area of Science:
- Molecular Biology
- Structural Biology
- RNA Processing
Background:
- The PWI motif is a conserved domain found in SRm160, a splicing and 3'-end cleavage-stimulatory factor.
- Its function and role in pre-mRNA processing were previously unknown.
Purpose of the Study:
- To characterize the PWI motif as a novel nucleic acid-binding domain.
- To elucidate its structural basis for RNA and DNA binding.
- To understand its contribution to SRm160 function in 3'-end pre-mRNA processing.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to determine the solution structure of the SRm160-PWI motif.
- Site-directed mutagenesis to investigate structure-function relationships.
- Biochemical assays to assess RNA/DNA binding and stimulation of 3'-end cleavage.
Main Results:
- The PWI motif is a novel alpha-helical fold (four-helix bundle) capable of binding both single- and double-stranded nucleic acids.
- Deletion of the PWI motif abolishes SRm160's RNA-binding and 3'-end cleavage stimulation.
- Structure-guided mutagenesis identified a conserved surface and adjacent basic region critical for nucleic acid binding.
Conclusions:
- The PWI motif represents a new class of nucleic acid-binding domain.
- It plays a vital role in SRm160-mediated stimulation of 3'-end pre-mRNA formation.
- This domain likely has broader implications in other pre-mRNA processing factors.