A U1-U2 snRNP Interaction Network during Intron Definition

Abstract

The assembly of prespliceosomes is responsible for selection of intron sites for splicing. U1 and U2 snRNPs recognize 5= splice sites and branch sites, respectively; although there is information regarding the composition of these complexes, little is known about interaction among the components or between the two snRNPs. Here we describe the protein network of interactions linking U1 and U2 snRNPs with the ATPase Prp5, important for branch site recognition and fidelity during the first steps of the reaction, using fission yeast Schizosaccharomyces pombe. The U1 snRNP core protein U1A binds to a novel SR-like protein, Rsd1, which has homologs implicated in transcription. Rsd1 also contacts S. pombe Prp5 (SpPrp5), mediated by SR-like domains in both proteins. SpPrp5 then contacts U2 snRNP through SF3b, mediated by a conserved DPLD motif in Prp5. We show that mutations in this motif have consequences not only in vitro (defects in prespliceosome formation) but also in vivo, yielding intron retention and exon skipping defects in fission yeast and altered intron recognition in budding yeast Saccharomyces cerevisiae, indicating that the U1-U2 network provides critical, evolutionarily conserved contacts during intron definition. Intron removal from new transcripts by pre-mRNA splicing is a fundamental feature of all eukaryotes. Such splicing is catalyzed by the spliceosome, a dynamic RNA-protein complex containing Ͼ150 proteins and five snRNAs. The assembly of the spliceosome is considered to be a dynamic process with a large number of RNA-RNA and RNA-protein rearrangements (31, 35). In the canonical pathway, U1 snRNP recognizes the pre-mRNA at the 5= splice site (5=SS); then, U2 snRNP stably binds the branch site (BS) region to form a prespliceosome. U4/5/6 tri-snRNP then joins, after rearrangements, U1 and U4 snRNPs are released, and the remaining U2/5/6 core forms the catalytic spliceosome. Both the early recognition of pre-mRNA and the rearrangements of snRNP structures to form an active conformation are facilitated by DExD/H ATPases, which couple ATP binding/hydrolysis with structural alterations (33,35).Two modes for early exon and intron specification have been described: exon definition for short exons flanked by long introns, which mostly appear in vertebrates, and intron definition for short introns, which are often present in lower eukaryotes (5). Interactions between U1 and U2 snRNPs are critical to both exonand intron-defined phases of spliceosome assembly. In the formation of commitment complexes in budding yeast Saccharomyces cerevisiae, or E complexes in mammals, cross-intron bridging interactions are proposed to connect from Prp40 in U1 snRNP at the 5=SS to SF1/BBP at the branch site or to U2AF at the polypyrimidine tract (PPT), respectively (1, 24). However, in prespliceosome formation, the first ATP-dependent transition, the BS-SF1/BBP interaction (or the PPT-U2AF interaction) is disrupted and replaced by BS-U2 snRNP interactions (28,33). This exchange of interactions is facilitated by the ATPase Prp...

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

“…S2 with Xu and Query 2007). We previously observed in fission yeast S. pombe that Prp5p binds U2 snRNP through the SF3B complex (Xu et al 2004;Shao et al 2012). Here, we show that Hsh155p/SF3B1 HEAT repeats are the interface for the Prp5-U2 snRNP interaction (Fig.…”

“…In addition, we previously identified prp5-DPLD mutant alleles in the conserved DPLD motif at the N terminus of Prp5p to improve splicing of BS mutant reporters (Shao et al 2012). In vitro protein-protein interaction assays, as above, revealed that Prp5-DPLD-to-APLD and Prp5-DPLD-to-AAAA mutant proteins pulled down more Hsh155 protein, including HEAT 1-8, 5-12, and 9-16 fragments, at constantly mild levels (Fig.…”

“…Competition between the ATPase activity of Prp5p and the stability of the BS-U2 snRNA duplex impacts branch region fidelity: Alteration of Prp5p's ATPase activity can increase (slower ATPase) or decrease (faster ATPase) use of suboptimal BSs (Xu and Query 2007;Shao et al 2012;. hsh155 alleles affect splicing of exactly the same suboptimal BS-U2 pairings as prp5 alleles (cf.…”

“…The conformational changes that occur during spliceosomal assembly, besides being dependent on the action of nucleic acid-dependent ATPases, confer kinetic proofreading for splice site selection (Wahl et al 2009;Semlow et al 2016). The early entry of U2 snRNP in the spliceosomal assembly process and its interaction with Prp5 (Xu and Query 2007;Shao et al 2012), an ATPase essential for prespliceosomal formation and fidelity, lead us to hypothesize that the consequences of SF3B1 disease-related mutations were at this stage.…”

“…Prp5 interacts with U1 snRNP through its N-terminal SR-like domain and with the U2 snRNP SF3B complex through its DPLD motif, providing a U1:Prp5:U2 platform for prespliceosome formation (Xu et al 2004;Shao et al 2012). Mutations in both the ATPase domain and the DPLD motif of Prp5 modulate splicing fidelity, specifically favoring the recognition and use of suboptimal substrates at the BS region (Xu and Query 2007;Liang and Cheng 2015).…”