Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing.
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.
Silverman
Edward J
EJ
City of Hope Graduate School of Biological Sciences, Duarte, CA 91010, USA.
Maeda
Ayaka
A
Wei
Janet
J
Smith
Paul
P
Beggs
Jean D
JD
Lin
Ren-Jang
RJ
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000483:Alleles; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D053487:DEAD-box RNA Helicases; D017930:Genes, Reporter; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012326:RNA Splicing; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
C096994:SPP2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D012313:RNA; D005982:Glutathione Transferase; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D005998:Glycine