Purification and characterization of several components of the RNA editing machinery from Leishmania tarentolae mitochondria: Ribonucleoprotein complexes, guide RNA-binding proteins and terminal uridylyl transferase.

摘要

The mitochondrial mRNA of kinetoplastid protozoa is posttranscriptionally modified by the insertion or deletion of the uridine residues, thus establishing or correcting the reading frame of the message. This process is mediated by small antisense transcripts encoded in the mitochondrial genome, termed gRNAs. The 5;Several RNP complexes have been identified that may be involved in RNA editing. When the mitochondrial extract of Leishmania tarentolae was incubated with labeled UTP and separated by native gel electrophoresis, six so-called "T-complexes" were visualized. It was determined that the T-IV complex contains gRNA and terminal uridylyl transferase (TUTase). An RNA ligase activity was identified by dimerization of a small synthetic RNA and the presence of two labeled polypeptides, 45 and 50 kDa, that may represent the adenylated intermediates of RNA ligase. The ligase activity sedimented at 20S and coincided with an gRNA-independent U-incorporation activity. An ATP-labeled complex was identified in the 20S fraction by native gel electrophoresis.;A fractionation scheme has been developed to purify gRNA-binding proteins and TUTase from mitochondrial extract. Twelve gRNA-binding proteins have been identified and partially characterized. TUTase has been enriched 7200 fold. Two candidate polypeptides, 140 kDa and 130 kDa copurify with TUTase activity. The partially purified TUTase adds UMP residues to the 3