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.
Highlights
Alternative splicing in metazoans produces multiple mRNA transcripts from a single gene and is a powerful mechanism for amplifying proteome complexity
In addition to its nuclear splicing activity PTB is found in the cytoplasm where it has roles in the stabilisation and localization of mRNA (Ghetti et al, 1992; Cote et al, 1999; Tillmar et al, 2002); it is recruited to stimulate translation initiation driven by internal ribosome entry sites (IRES) from cellular and viral mRNAs (Sawicka et al, 2008)
The crystal structures of PRI3 and PRI4 from Raver1 in complex with PTB RRM2 are consistent with many of the features found in previous work: the peptides bind to the dorsal helical face of the RNA recognition motif domains (RRMs) domain and in the same orientation as determined by NMR methods (Fig. 1B) (Rideau et al, 2006)
Summary
The polypyrimidine tract binding protein (PTB) is an important regulator of alternative splicing. PTBregulated splicing of α-tropomyosin is enhanced by Raver, a protein with four PTB-Raver Interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. High-affinity binding by Raver 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-Raver interactions.
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