Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these func tions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Pr eviously, we have characterized the glycolytic enzyme glyceraldehyde-3-phos phate dehydrogenase (GAPDH) as an RNA-binding protein with preference to ad enine-uracil-rich sequences. In this study, we used GST-GAPDH fusion protei ns generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which c orrespond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that thi s single domain, although it retains most of the RNA-binding activity, lose s sequence specificity. Our results suggest a molecular basis for RNA-recog nition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabo lic enzymes that had been reported to have RNA-binding activity with differ ent specificity. To support this prediction we also identified other member s of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our fin dings we propose the addition of the NAD(+)-binding domain to the list of R NA binding domains/motifs. (C) 2000 Academic Press.