MITOCHONDRIAL GLUTAMATE-DEHYDROGENASE FROM LEISHMANIA-TARENTOLAE IS AGUIDE RNA-BINDING PROTEIN

To identify specific proteins interacting with guide RNAs (gRNAs) in m itochondrial ribonucleoprotein complexes from Leishmania tarentolae, f ractionated and unfractionated mitochondrial extracts were subjected t o UV cross-linking with added labeled gRNA and also with [alpha-P-32]U TP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localize d in the T-V complex, which sediments in glycerol gradients at the lea ding edge of the 10S terminal uridylyltransferase peak was found to in teract with both types of labeled RNAs. The p110 protein was gel isola ted and subjected to microsequence analysis, and the gene was cloned, The sequence revealed significant similarity with mitochondrial glutam ate dehydrogenases. A polyclonal antiserum was raised against a recomb inant fragment of the p110 gene and was used to demonstrate a stable a nd specific gRNA-binding activity by coimmunoprecipitation and competi tive gel shift analyses. Complex formation was strongly inhibited by c ompetition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated tr anscript was sufficient for p110 binding. Both the gRNA-binding activi ty of the p110 protein and in vitro gRNA-independent and gRNA-dependen t uridine insertion activities in the mitochondrial extract were inhib ited by high concentrations of dinucleotides.