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.
Highlights
Yeast pre-mRNA splicing (1–3) starts with the assembly of a complex comprising the U1 snRNP bound at the intron 5 splice site and the Msl5Mud[2] heterodimer engaged at the intron branchpoint
Dissociation of U1 snRNP is thought to be triggered by the DEAD-box protein Prp[28] (4,5), acting to disrupt the short U1:5 splice site RNA duplex or remodel protein–RNA contacts at the 5 splice site
This hypothesis regarding Prp[28] function is predicated on findings that the essentiality of Prp[28] for vegetative growth can be bypassed by mutations in the essential U1 snRNP subunits Yhc[1], Prp[42], Snu[71] and SmD3 and by specific U1 snRNA mutations located within and flanking the segment that base-pairs with the intron 5 splice site (4,6–8). These genetic interactions imply that the need for Prp[28] during U1 snRNP ejection from the early spliceosome is alleviated by certain alterations that weaken U15 splice site contacts
Summary
Yeast pre-mRNA splicing (1–3) starts with the assembly of a complex comprising the U1 snRNP bound at the intron 5 splice site and the Msl5Mud[2] heterodimer engaged at the intron branchpoint. These genetic interactions imply that the need for Prp[28] during U1 snRNP ejection from the early spliceosome is alleviated by certain alterations that weaken U15 splice site contacts
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