Interaction between a G-Patch Protein and a Spliceosomal DEXD/H-Box ATPase That Is Critical for Splicing

Abstract

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/ H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.Pre-mRNA splicing is a dynamic process through which snRNPs and trans-acting proteins interact in an ordered manner in the spliceosome to remove introns from pre-mRNA. The process of splicing can be broken down in vitro to the steps of spliceosome formation, the first transesterification reaction, the second transesterification reaction, the release of mature mRNA, and the recycling of the snRNPs. DEXD/H-box helicases act upon the spliceosome to catalyze assembly, activation, and disassembly of the spliceosome in an ordered, stepwise manner (40). Comprehending the action of these helicases and their effect on the spliceosome is essential to understanding the process of pre-mRNA splicing.Prp2 is an RNA-dependent ATPase (21) belonging to the DEAH subfamily of DEXD/H-box helicases that includes Prp16, Prp22, and Prp43 (40). The DEAH splicing factors share homology extending from the helicase domain through the carboxyl terminus. Despite the extensive homology shared between the DEAH family splicing helicases, each has a distinct, sequential role in the splicing reaction. Prp2 activates the spliceosome upon ATP hydrolysis before the first transesterification reaction (20, 22), Prp16 acts before the second transesterification reaction (38), Prp22 has roles in the second step of pre-mRNA splicing and mRNA release from the spliceosome (37, 42), and Prp43 is involv...

Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function

“…Mutational studies suggest that the G-patch element in Spp382p is needed to stimulate Prp43p RNA helicase activity (Pandit et al 2006;Tanaka et al 2007) and in Spp2p to recruit Prp2p to the spliceosome (Silverman et al 2004). While these observations suggest a proteinrecognition interface, the G-patch motif has also been proposed to serve as an RNA-binding function (Bauerova-Zabranska et al 2005) and such function has not been ruled out for any of the yeast G-patch proteins.…”

“…Of the five G-patch proteins annotated in the MIPS Comprehensive Yeast Genome Database (http:/ /mips.gsf.de/ genre/proj/yeast/), three are essential (Spp2p, Spp382p) or nearly essential (Pxr1p) and assist DExD/ H-box factors in RNA processing. Spp2p functions with Prp2p in splicing (Silverman et al 2004) while Spp382p and Pxr1p act with Prp43p in splicing and ribosome biogenesis, respectively (Guglielmi and Werner 2002;Tsai et al 2005;Boon et al 2006;Pandit et al 2006;Tanaka et al 2007). Pxr1p and Spp382p also influence telomerase activity and genomic stability (Lin and Blackburn 2004;Herrmann et al 2007), suggesting additional contributions to nuclear function.…”

Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae

“…Lysine residues within this domain can be chemically cross-linked with the Spp382 G-patch consistent with close and perhaps direct contact between these peptide features (Christian et al 2014). Similar interactions are predicted to occur between the C-terminus of the Prp2 DEAHbox helicase and its G-patch activator, Spp2 (Roy et al 1995;Silverman et al 2004;Warkocki et al 2015). It was surprising, therefore, that the large Prp43 C-terminal domain (CTD) deletion scored here had no discernible impact on G-patch-dependent Spp382-Prp43 Y2H interaction.…”

The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome

“…Like most other spliceosomal DExD/H-box ATPases, the association of Prp2p with the spliceosome is transient (King and Beggs 1990). When Prp2p activity is required during spliceosome activation, Prp2p is recruited to the spliceosome through its interaction with the G-patch protein Spp2 and then, after ATP hydrolysis by Prp2p, both factors dissociate (Roy et al 1995;Kim and Lin 1996;Silverman et al 2004;Fabrizio et al 2009). Like many other DExD/H-box ATPases, the ATPase activity of Prp2p is stimulated by RNA binding (Kim et al 1992).…”