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Import of small RNAs into Leishmania mitochondria in vitro
S Mahapatra1, T Ghosh, S Adhya
1Genetic Engineering Laboratory, Indian Institute of Chemical Biology, Calcutta.
Abstract:
Using an in vitro ribonuclease protection assay, it was shown that synthetic antisense transcripts from the 5'-upstream region of the beta-tubulin gene are efficiently imported into isolated Leishmania mitochondria. 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 rapid ATP-dependent uptake. Import was saturable with antisense RNA at about 1 nM concentration, and sequence-specific, as shown by lack of import 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) and Leishmania tRNA competed with antisense RNA for import. Incubation of mitochondria with srRNAs and tRNA in the presence of radiolabelled UTP resulted in the ribonuclease-resistant labelling of these RNAs by the mitochondrial terminal uridylyl transferase. Extracts of isolated mitochondria contain a factor binding to antisense RNA, as shown by gel retardation assay. These observations indicate the presence of a receptor-mediated import pathway for srRNAs and tRNA in Leishmania mitochondria.
Insights
Leishmania mitochondria import antisense RNA via a receptor-mediated pathway, dependent on ATP and specific RNA sequences. This process involves initial RNA-mitochondria interaction followed by uptake, suggesting a novel import mechanism for mitochondrial RNAs.
Area of Science:
- Mitochondrial biology
- Molecular genetics
- Parasitology
Background:
- Mitochondrial gene expression regulation is complex.
- RNA import into mitochondria is a poorly understood process.
- Leishmania mitochondria possess unique features.
Purpose of the Study:
- To investigate the mechanism of RNA import into Leishmania mitochondria.
- To identify factors and characteristics of the RNA import pathway.
- To determine if antisense transcripts can be imported.
Main Methods:
- In vitro ribonuclease protection assay.
- ATP-dependent import assays.
- Competition assays with other RNAs.
- Gel retardation assays to detect RNA-binding factors.
Main Results:
- Antisense transcripts from the beta-tubulin gene are imported into Leishmania mitochondria.
- Import is ATP-dependent, saturable, and sequence-specific.
- Oligopurine motifs on antisense RNA correlate with import efficiency.
- Small ribosomal RNAs and tRNA compete for import, suggesting a shared pathway.
- A mitochondrial factor binds to antisense RNA.
Conclusions:
- Leishmania mitochondria possess a receptor-mediated import pathway for specific RNAs.
- This pathway likely involves small ribosomal RNAs and tRNA.
- The findings provide insights into mitochondrial RNA import mechanisms in parasites.