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

Abstract

A substantial amount of cytochrome oxidase subunit HI (COHI) mRNA continues to be synthesized de novo in Trypanosoma brucei in the presence of actinomycin D, presumably by a DNA-independent transcription process. We describe the identification of negative-strand corn RNA molecules, characterization of their termini, and the detection of RNA-dependent RNA polymerase activity. Three lines of evidence for the existence of negative-strand corn RNA are presented: (i) hybridization with oligonucleotide probes with the same polarity as mRNA after preliminary enrichment for putative negative-strand RNA by affinity purification; (ii) cloning and sequencing of negative-strand complements for the unedited, edited, and partially edited corn RNA; and (iii) exact correspondence of the terminal sequences of the putative negative-strand RNA molecules to the ends of con RNA. The presence of negative-strand complements of coM RNA is consistent with the notion that a significant amount of mRNA in T. brucei is synthesized by an RNA-dependent RNA polymerase with negative-strand RNA as an intermediate template.RNA metabolism in trypanosomes has a number of unusual aspects. Two novel reactions were first discovered in these organisms-trans-splicing and RNA editing. Trans-splicing, the processing of pre-mRNA by joining of independent RNA transcripts, accounts for the presence of an identical 39-nucleotide sequence at the 5' ends of all nuclear mRNAs in trypanosomes where the regular, cis-splicing is not observed (1, 2). RNA editing, which occurs in kinetoplast mitochondria, involves addition or occasionally the deletion of uridylate residues (3-7). This process occurs posttranscriptionally; and as a result the nucleotide sequence of the mature edited mRNA differs from that of the gene from which it is transcribed (8, 9).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. brucei of yet a third unusual feature of RNA metabolism, RNA-dependent RNA synthesis via negative-strand RNA intermediates. Here we describe the identification and characterization of negative-strand complements for cytochrome oxidase subunit III (COIII) RNA in procyclic T. brucei. MATERIALS AND METHODSCell Culture. The procyclic form of clone 570 of T. brucei, stock EVE10 (from I. Cunningham, University of Massachusetts, Amherst), was grown in Dulbecco's modified Eagle's medium with 20% fetal bovine serum.Isolation of RNA. Cells were lysed at 0C in 20 mM Hepes, pH 8.2/360 mM NaCl/4 mM MgCl2/8% (wt/vol) sucrose/ 1.5% (vol/vol) Triton X-100 containing RNasin (Promega) at 1000 units/ml. The cytoplasmic fraction was treated with proteinase K and extracted with phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol).RNA Electrophoresis, Blotting, and Hybridization. RNA samples were denatured and subjected to electrophoresis in 1.5% agaro...

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

“…A key prerequisite for this study was the development of procedures for reliably cloning antisense RNA and for the precise and reliable determination of the structure of its termini. We developed a multistep procedure to fulfill these objectives (4,5). Its initial step is the attachment to cellular RNA of a molecular tag.…”

“…In a typical ligation reaction, 10 ,ug of such RNA was mixed with 1 jig of ribooligo 1 that was pretreated with sodium periodate (8), CIP, and T4 polynucleotide kinase (4,5). Cellular RNA and ribooligonucleotide were heated at 100°C for 2 min in 1 mM EDTA (pH 6.2), mixed with dimethyl sulfoxide, and cooled on ice prior to addition of the rest of the components.…”

“…Hybridization with antisense globin RNA-specific probe provided an initial indication of the presence of these molecules in mouse erythroid tissues. The definitive identification, sequencing, and the determination of the termini of the antisense globin RNA were made possible by the development of ligation-mediated RNA amplification (4,5), which allowed the detailed analysis of this molecule.…”

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“…In other examples such as Drosophila melanogaster, cuticle protein (Henikoff et al 1986) and decarboxylase (Spencer et al 1986) genes, mouse tissue culture antisense transcripts (Williams & Fried 1986), cytochrome oxidase subunit III of Trypanosoma brucei (Volloch et al 1991), the mouse globin gene (Volloch et al 1996) and insulin-like growth factor-II (Sussenbach 1989), the role of antisense RNA transcription is unknown. Figure 4 Northern blots of mRNA from human tissues.…”

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