Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing

Abstract

SummaryThe polypyrimidine tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which—along with structure-based mutagenesis—reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.

Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2

“…4). Flag-tagged RRM2 from PTBP1 pulled down substantial amounts of Raver1, confirming the earlier results (Rideau et al 2006;Joshi et al 2011). However, RRM2 from PTBP2 showed a much weaker interaction, with the level of Raver1 in the Flag-IP slightly above background.…”

“…RNA recognition motifs (RRMs) are shaded gray (Oberstrass et al 2005). PTBP1.4 residues that interact with Raver1 are shaded light pink (Joshi et al 2011). Vertical lines above the sequence indicate PTBP1.4 residues that interact with RNA (Oberstrass et al 2005).…”

“…When assayed in nuclear extracts the proteins differed in their assembly with other factors Markovtsov et al 2000). On some introns, PTBP1 interacts with the RNA binding proteins Raver1/2, Matrin 3, or MBNL to form higher-order RNP complexes (Gromak et al 2003;Henneberg et al 2010;Joshi et al 2011;Gooding et al 2013;Coelho et al 2015). Of these, the PTBP1-Raver1 interaction is best characterized; the α helical surface of RRM2 interacts with a peptide motif within Raver1.…”

“…Of these, the PTBP1-Raver1 interaction is best characterized; the α helical surface of RRM2 interacts with a peptide motif within Raver1. PTBP2 was shown to bind this Raver1 peptide, but its interactions with full-length Raver1, or with Matrin 3 and MBNL are not known (Joshi et al 2011). These studies indicate that there are differences in how PTBP1 and PTBP2 interact with cofactors.…”

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…One of the first RRM structures revealed distinct α-helical and β-sheet surfaces of the U2B ′′ RRM bound to the U2A ′ protein and the U2 small nuclear (sn) RNA (Price et al 1998). Subsequent structures, including the complex of alternative splicing factors PTB with Raver1 (Rideau et al 2006;Joshi et al 2011), intramolecular CPEB1 contacts for polyadenylation (Afroz et al 2014), and the Snu17p RRM bound to Bud13p in the pre-mRNA retention and splicing complex (RES) (Tripsianes et al 2014;Yan et al 2016), establish that separate α-helical and RNP surfaces of the RRMs often bind protein and RNA partners simultaneously.…”