A conserved peptide motif in Raver2 mediates its interaction with the polypyrimidine tract-binding protein.
Raver2 was originally identified as a member of the hnRNP family through database searches revealing three N-terminal RNA recognition motifs (RRMs) bearing highest sequence identity in the RNP sequences to the related hnRNP Raver1. Outside the RRM region, both Raver proteins are quite divergent in sequence except for conserved peptide motifs of the [S/G][I/L]LGxxP consensus sequence. The latter have been implicated in Raver1 binding to the polypyrimidine tract-binding protein (PTB) a regulatory splicing repressor and common ligand of both Raver proteins. In the present study we investigated the association of Raver2 with RNA and PTB in more detail. The isolated RRM domain of Raver2 weakly interacted with ribonucleotides, but the full-length protein failed to directly bind to RNA in vitro. However, trimeric complexes with RNA were formed via binding to PTB. Raver2 harbors two putative PTB binding sequences in the C-terminal half of the protein, whose influence on Raver2-PTB complex formation was analyzed in a mutational approach, replacing critical leucine residues with alanines. While mutation of either sequence motif alone negatively affected Raver2 binding to PTB in vitro, only mutation of the more C-terminally located SLLGEPP motif significantly reduced the recruitment of Raver2 into perinucleolar compartments (PNCs) in HeLa cells. The latter observation was also confirmed for Raver1: out of four sequence motifs matching the PTB binding consensus, mutations in the SLLGEPP motif were the only ones attenuating the recruitment of Raver1 into PNCs. The conserved mode of PTB binding suggests that Raver2, like Raver1, may function as a modulator of PTB activity.
Henneberg
Berenike
B
Zoological Institute, Technical University of Braunschweig, D-38106 Braunschweig, Germany.
Swiniarski
Sascha
S
Sabine Becke
Illenberger
Susanne
S
D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D010455:Peptides; D016601:RNA-Binding Proteins; C442838:Raver1 protein, mouse; C503919:Raver2 protein, mouse; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA