Abstract
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 StNova1, was identified as a potential interacting partner of the StPTB 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 StNova1 and StPTB1 and −6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins StPTB1/6 and StNova1 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 StPTB 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 StNova1. We show, through site-specific mutations, the importance of glycine and proline residues in StNova1-StPTB interactions.
Highlights
Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that have been well characterized and studied in vertebrates. They typically contain four RNA recognition motifs (RRM) that are separated by three linker regions and all the RRM domains have been shown to be involved in RNA binding
The RRMs interact with sequence in UTRs containing groups of four cytosine/uracil motifs at least four nucleotides in length [1,2].The PTBs participate in multiple regulatory functions in mRNA metabolism including polyadenylation, 39 end formation, translation from internal ribosomal entry sites, RNA localization and stability, and alternative splicing (AS) [2,3,4]
To identify potential proteins interacting with StPTB proteins we performed a yeast two-hybrid screening against leaf proteins using StPTB1 as bait
Summary
Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that have been well characterized and studied in vertebrates. The Nova1-like protein of Arabidopsis thaliana, designated BTR1 (binding to ToMV RNA 1L), binds to terminal regions of genomic RNA of the tomato mosaic virus (ToMV) [12]. These terminal regions contain important regulatory elements for translation and RNA replication. The Nova-1 family of genes is highly conserved within the plant kingdom with orthologs in numerous diverse species, including Glycine max, Hordeum vulgare, Medicago truncatula, Oryza sativa, Populus trichocarpa, Ricinus communis, Solanum lycopersicum, Vitis vinifera, and Zea mays Despite this conservation, with the exception of BTR1, very little is known about the function of these plant orthologs
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