Mapping and Characterization of the Interaction Interface between Two Polypyrimidine-Tract Binding Proteins and a Nova-Type Protein of Solanum tuberosum

Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length PTB proteins have been identified. In the present study Nova1-like protein, designated St Nova1, was identified as a potential interacting partner of the St PTB proteins via yeast two-hybrid screening. Nova protein is a RNA-binding protein that contains three K-homology (KH) domains. In humans, these proteins are involved in regulation of neuronal RNA metabolism but the role of Nova-like proteins in plants is poorly understood. We have validated this interaction and mapped the protein binding region on St Nova1 and St PTB1 and −6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins St PTB1/6 and St Nova1 is mediated through linker regions that are distinctly separated from the RRMs. Furthermore, using a random 21-mer phage-peptide library, we have identified a number of peptides with the consensus sequence motif [S/G][V/I][L/V]G that recognize the St PTB proteins. One over-represented peptide that recognizes StPTB6 contains the GVLGPWP sequence that is similar to the GIGGRYP sequence in the glycine-rich linker region between the KH2 and KH3 domains of St Nova1. We show, through site-specific mutations, the importance of glycine and proline residues in St Nova1- St PTB interactions.