Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei

Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain. Components of this complex represent a form of post-transcriptional operon because trypanosome mitochondrial activity is unusual in being developmentally programmed. By genome analysis we identified the genes for seven components of the COX complex. Each mRNA exhibits bloodstream stage-specific instability, which is not mediated by the RNA silencing pathway but which is alleviated by cycloheximide. Reporter assays have identified regulatory regions within the 30-untranslated regions of three COX mRNAs operating principally at the translational level, but also via mRNA stability. Interrogation of the mapped regions via oligonucleotide frequency scoring provides evidence for genome-wide conservation of regulatory sequences among a large cohort of procyclic-enriched transcripts. Analysis of the coregulated subunits of a stage-specific enzyme is therefore a novel approach to uncover cryptic regulatory sequences controlling gene expression at the post-transcriptional level. INTRODUCTION The analysis of cohorts of mRNAs involved in functionally co-ordinated processes is providing a useful tool for the identification of regulatory signals controlling gene expression in eukaryotes (1). Of particular value has been the characterization of the regulatory signals and RNA binding proteins that control nuclear-encoded genes responsible for mitochondrial function. For example, in Saccharomyces cerevisiae, mitochondrial function is regulated dynamically in response to a shift between a fermentable and non-fermentable carbon source, oxygen and haem. This has enabled the experimental analysis (2) and computational prediction (3,4) of cryptic regulatory motifs governing expression. The requirement to identify control elements in mRNAs which are subject to regulated expression is particularly acute in the parasite Trypanosoma brucei. These organisms, the causative agents of human and animal African trypanosomiasis, are evolutionarily divergent protozoa that are transmitted between mammalian hosts by the tsetse fly (Glossina spp) (5). The different environments that the parasite encounters during its complex life-cycle represent extreme challenges that the cell must overcome, this necessitating regulation of such fundamental processes as metabolism, cell morphology and cell cycle control (6). Unusually, however, these processes are not controlled by regulated transcription. This is because the genome is organized into polycistronic transcription units in which co-transcribed genes are not necessarily co-regulated (7). This genome organization requires resolution of polycistronic premRNAs via the RNA processing reactions of trans-splicing and polyadenylation, generating mRNAs with a 39 nt 50 spliced leader sequence and 30 poly(A) tail. Interestingly, trans-splicing and polyadenylation appear to be mechanistically coupled, with the polyadenylation of the upstream gene in an array being co-ordinated with trans-splicing for the downstream gene (8,9). This linkage of RNA processing *To whom correspondence should be addressed. Tel: +44 131 651 3639; Fax: +44 131 651 3670; Email: keith.matthews@ed.ac. uk