A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified proteinprotein interactions

More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination invitro or in a mouse. What, then, is the function of the N-terminal domain ? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional proteinprotein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1 and found two othersto the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to proteinprotein interactions that may also influence how RAG1 binds DNA targets.