Abstract
The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis. The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors. This domain promotes the homodimerization of protein L7 through alpha-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation. Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7. A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions. Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 x 10(-9) M) and, respectively, ZNF7-L7 (2 x 10(-9) M) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody. We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form. This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.
Highlights
We have used the yeast two-hybrid system to identify from a Jurkat T lymphoma cDNA library ribosomal protein S7 and multi-zinc finger protein ZNF7 as proteins that interact with ribosomal protein L7
Ribosomal protein L7 is located at the surface of the large 60 S ribosomal subunit [30, 31], to which it associates in the cytoplasm [32]
The N-terminal basic region leucine zipper (BZIP)-like domain of L7 is thought to be exposed to the cytoplasm due to its ability to bind mRNAs with high affinity [3, 4] and because of crosslinking and labeling experiments [31, 33]
Summary
Standard Techniques—Manipulations of Escherichia coli, Saccharomyces cerevisiae, nucleic acids, and proteins were performed as described [16]. Specificity of Interaction—Rescued library plasmids of S7 and ZNF791–687 were retransformed into EGY48/pJK103, containing as baits either pRFHM1 [15], which encodes the homeodomain of bicoid, or pLexA-myc [19], which encodes the carboxyl-terminal 176 amino acids of human c-Myc. Transformants were assayed for leu and lacZ activity. Antibodies against HisZNF791–687 and against the ovalbumin-coupled peptide sequence CGRIHTGEKPYKG (anti-ZNF7PEP) that represents the conserved spacer sequence between zinc finger domains in ZNF7 and many other human zinc finger proteins were raised in rabbits. Both antisera recognized on immunoblots of cell lysates a band with the predicted electrophoretic mobility of ZNF7 (78 kDa) and two additional bands of 55 and 57 kDa, respectively. Universitat Konstanz, Konstanz, FRG), which were treated as described above
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