IMPORT OF SMALL RNAS INTO LEISHMANIA-MITOCHONDRIA IN-VITRO

Using an in vitro ribonuclease protection assay, it was shown that syn thetic antisense transcripts from the 5'-upstream region of the beta-t ubulin gene are efficiently imported into isolated Leishmania mitochon dria. Import occurred after a lag of about 30 min at 25 degrees C and was dependent on ATP. Preincubation experiments suggested that import consists of a slow interaction of mitochondria with RNA, followed by r apid ATP-dependent uptake. Import was saturable with antisense RNA at about 1 nM concentration, and sequence-specific, as shown by lack of i mport of other labelled transcripts. Deletion analysis demonstrated a correlation between efficiency of import and the number of oligopurine motifs on the antisense RNA. Several small ribosomal RNAs (srRNAs) an d Leishmania tRNA competed with antisense RNA for import. Incubation o f mitochondria with srRNAs and tRNA in the presence of radiolabelled U TP resulted in the ribonuclease-resistant labelling of these RNAs by t he mitochondrial terminal uridylyl transferase. Extracts of isolated m itochondria contain a factor binding to antisense RNA, as shown by gel retardation assay. These observations indicate the presence of a rece ptor-mediated import pathway for srRNAs and tRNA in Leishmania mitocho ndria.