Uncoupling of the synthesis of edited and unedited COIII RNA in Trypanosoma brucei

Abstract

RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.

Spliced leader RNA trans-splicing in dinoflagellates

“…Thus, all queries for the presence of SL in organellar transcripts were negative (SI Table 1). Similarly, no product was obtained in PCRs that used DinoSL and small subunit ribosomal RNA (18S rRNA)-specific primer sets for cDNA synthesized with random hexamers, indicating that SL is not added to 18S rRNA, a result consistent with previous reports that SL is not present at the 5Ј end of the transcripts of 5.8S rRNA (i.e., RNA pol I) (21), 5S rRNA (i.e., RNA pol III) (22), or mitochondrial mRNAs in Trypanosoma brucei (23), although trans-splicing of the geneinternal variety occurs in mitochondrial genes of some organisms (24). The presence of SL on nuclear-encoded mRNAs of proteins destined for organellar import supports the hypothesis that transsplicing is generally required for nuclear gene expression; alternatively, adoption of the trans-splicing pathway may have allowed an easier transition for gene relocation into the nuclear genome.…”

Identification of negative-strand complements to cytochrome oxidase subunit III RNA in Trypanosoma brucei.

“…On the other hand, the complementarity of positive-and negative-strand COIII RNA, including their termini, and the complementary disposition of edited regions in partially edited positive-and negative-strand RNA molecules suggest a template-product relationship between positive-and negative-strand RNA. This structural relationship, taken together with the demonstration that a significant proportion of edited COIII RNA is synthesized as a unit (10) and the identification of an RNA-dependent RNA polymerase activity in extracts of T. brucei (V.V., B.S., and S.R., unpublished results), is consistent with the function of negative-strand RNA as an intermediate template in RNAdependent RNA synthesis. Such a process, which seems to have no obvious relationship to the process of RNA editing, may play an important role in RNA metabolism in T. brucei as a mechanism of RNA amplification.…”

“…In the course of studies of RNA editing, we observed that a significant proportion of some edited mRNA species in Trypanosoma brucei is the product of an actinomycin D-resistant (i.e., presumably DNA-independent) transcriptional process (10). This finding suggested the occurrence in T.…”

“…RNA samples were denatured and subjected to electrophoresis in 1.5% agarose gels containing formamide and formaldehyde (10). Blots were hybridized in the presence of nonspecific oligonucleotide (200 ,ug/ml) of size comparable to the size of the probe.…”

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

“…For gel analysis, RNA samples (30 ,ug) were denatured (3 min at 100°C in 1 mM EDTA, pH 6.2), resolved in 1.5% agarose/formaldehyde gels, blotted to nitrocellulose, and hybridized with 5'-end-labeled oligonucleotides specific for either sense (probe 1) or antisense (probe 2) globin RNA as described (7). The blot hybridized with sense strand-specific probe was exposed for 45 min, whereas the blot hybridized with antisense strand-specific probe was exposed for 3 days.…”