A Cis-acting A-U Sequence Element Induces Kinetoplastid U-insertions

Abstract

A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolae cytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5 and 3 of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing.

Activation of Guide RNA-directed Editing of a Cytochromeb mRNA

“…Without addition of gRNA to the editing reaction, only the artifact primer extension bands are visible (Fig. 2B, lane 2), consistent with previous results indicating that gRNA-independent insertions do not occur to a significant extent at the first editing site (9).…”

“…These insertions do not create a functional coding sequence, and their relevance to the gRNA-directed reaction remains unclear. Intermediates produced during the gRNA-independent reaction, however, are consistent with the cleavage/U-insertion/ligation mechanism proposed for the directed reaction (9). A 34-nucleotide untranslated A-U sequence element located immediately upstream of the cytochrome b editing sites was found to be necessary for the gRNA-independent U-insertions, and the majority of these U-insertions occur immediately 5Ј and 3Ј of the A-U sequence.…”

“…The culture was then diluted 3-fold in fresh media and grown to a density of 1.6 Ϯ 0.2 ϫ 10 8 cells/ml, at which point the cells were harvested and mitochondria prepared as described previously (9). The mitochondria were resuspended in 950 l of solubilization buffer (25 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EDTA, 20 mM KCl, 0.1 mM ATP, 1 mg/ml Pefabloc, 10 g/ml leupeptin, and either 10% glycerol or 10 mg/ml BSA) for every 500 ml of starting culture.…”

“…By using 4-thio-UTP in place of UTP in the editing reaction, RNAs containing insertions at editing sites could then be enriched by partitioning through an organomercury matrix (9). cDNA synthesis of the enriched RNA was primed with oligodeoxynucleotide 7534, and the cDNA was amplified with 7534 and oligodeoxynucleotide 190703.…”

“…1). The similarity in the two sequences suggested that it could also be involved in gRNA-directed editing, perhaps acting as a binding site for editing components (9). However, the lack of an in vitro system supporting gRNAdirected editing of cytochrome b made it difficult to assess its significance.…”