RAG-1 INTERACTS WITH THE REPEATED AMINO-ACID MOTIF OF THE HUMAN HOMOLOG OF THE YEAST PROTEIN SRP1

Genes for immunoglobulins and T-cell receptor are generated by a profe ss known as V(D)J recombination. This process is highly regulated and mediated by the recombination activating proteins RAG-1 and RAG-2. By the use of the two-hybrid protein interaction system, we isolated a hu man protein that specifically interacts with RAG-1. This protein is th e human homologue of the yeast SRP1 (suppressor of a temperature-sensi tive RNA polymerase I mutation). The SRP1-1 mutation is an allele-spec ific dominant suppressor of a temperature-sensitive mutation in the zi nc binding domain of the 190-kDa subunit of Saccharomyces cerevisiae R NA polymerase I. The human SRP cDNA clone was used to screen a mouse c DNA library. We obtained a 3.9-kbp cDNA clone encoding the mouse SRP1. The open reading frame of this cDNA encodes a 538-amino acid protein with eight degenerate repeats of 40-45 amino acids each. The mouse and human SRP1 are 98% identical, while the mouse and yeast SRP1 have 48% identity. After cotransfection of the genes encoding RAG-1 and human SRP1 into 293T cells, a stable complex was evident. Deletion analysis indicated that the region of the SRP1 protein interacting with RAG-1 i nvolved four repeats, The domain of RAG-1 that associates with SRP1 ma pped N-terminal to the zinc finger domain. Because this region of RAG- 1 is not required for recombination and SRP1 appears to be bound to th e nuclear envelope, we suggest that this interaction helps to localize RAG-1.