Developmental regulation of gene expression in trypanosomatid parasitic protozoa

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Kinetoplastids branched early from the eukaryotic lineage and include several parasitic protozoan species. Up to several hundred kinetoplastid genes are co-transcribed into polycistronic RNAs and individual mRNAs are resolved by coupled co-transcriptional trans-splicing of a universal splice-leader RNA (SL-RNA) and 3′-end maturation processes. Protein-coding genes lack RNA polymerase II promoters. Consequently, most of gene regulation in these organisms occurs post-transcriptionally. Over the last few years, many more genes that are regulated at the mRNA stability level and a few at the translation level have been reported. Almost all major trypanosome homologues of yeast/mammalian mRNA degradation enzymes have been functionally characterized and major pathways identified. Novel paradigms have also recently emerged: regulated post-transcriptional processing of cytoplasmic RNAs, SL-RNA transcriptional silencing-mediated global stress response, and Leishmania-specific large-scale modulation of post-transcriptional gene expression via inactive degenerated retroelements. Several of these developments have greatly benefited from the recently completed genomic sequences and functional genomic studies.

Introduction

The protozoan pathogens Trypanosoma brucei, T. cruzi and Leishmania spp. (family Trypanosomatidae, order kinetoplastida), referred to as TriTryps, diverged early on in the eukaryotic lineage and therefore display several unusual or unique biological features, some of which were subsequently found in other eukaryotes. Beyond their service as model organisms, these pathogens cause various diseases of high medical (sleeping sickness, Chagas disease and leishmaniasis, respectively) and veterinary importance that collectively affect millions of people worldwide, primarily in tropical and subtropical regions.

The life cycle of several trypanosomatid parasitic protozoa involves a mammalian host and an arthropod vector. African trypanosomes (T. brucei) live extracellularly both in the blood of mammals (e.g. bloodstream forms) and in the midgut of Tsetse flies (e.g. procyclics); T. cruzi reside as amastigotes in the cytosol of macrophages and as extracellular epimastigotes in the gut of triatomine bugs; Leishmania multiply as amastigotes within mononuclear phagocytes and as extracellular promastigotes in the sandfly vector. Drastic change in temperature between the poikilothermic insect vectors and homeothermic mammal is usually a shared challenge. Other environmental changes can be unique to the life style of a given parasite (e.g. the acidic pH that Leishmania face within the phagolysosomes of host macrophages). Some of these changes are known to constitute the stimuli that drive parasite differentiation programs and the parasites employ various adaptive strategies to survive and strive inside their distinct hosts.

Comparative genomics of TriTryps revealed that, more than half of their genes are well-conserved and map to highly syntenic regions [1•, 2, 3•]. Much of the species-specific adaptive strategies to different insect and life-cycle features, different target tissues, and distinct disease pathogenesis should, therefore, depend on protein sequence variations among orthologues, the non-syntenic part of their respective genomes and/or on highly regulated hierarchical changes in gene expression.

Global gene expression profiling during development and between species highlighted that several hundred genes are differentially regulated: 2–9% of all genes analyzed are modulated at the RNA level (presumably via mRNA stability; see below) and up to 12–18% are regulated at the protein level ([4•, 5, 6, 7], A. Rochette et al., unpublished). More proteins are found to be differentially expressed when the analysis included additional developmental forms of the parasites [8]. The difference between changes at the RNA and protein levels – the exact qualitative and quantitative nature of which is difficult to access because of inherent methodological limitations – is likely to be a function of translational and post-translational regulation (see below).

Genetic (e.g. gene amplification of the SL-RNA and drug resistance genes, and gene replacement of Variant Surface Glycoprotein genes (VSGs)) and ill-known epigenetic mechanisms of gene regulation are beyond the scope of this review. Here, the primary focus is on stage-specific post-transcriptional control, with emphasis on discoveries made in the past five years.

Section snippets

Polycistronic transcription and pre-mRNA processing

The recent completion of the TriTryp genomes indicates that protein-coding genes are organized as large bidirectional polycistronic units [1•, 9]. Transcription has been postulated to initiate at strand switch regions on each chromosome [9] in the absence of defined RNA pol II promoters and typical general transcription factors. The maturation of individual mRNAs from polycistronic pre-mRNAs involves two coupled co-transcriptional RNA-processing reactions: trans-splicing where a small capped

mRNA degradation and translation

Recent reviews have provided an exhaustive overview of our current knowledge regarding the processes of mRNA stability and translation in kinetoplastids [14•, 21, 22]. Degradation of eukaryotic mRNAs is typically initiated via deadenylation. In one pathway, deadenylation stimulates decapping and subsequent degradation of the mRNA body by 5′-exonucleases. In another pathway, deadenylation leads to degradation via 3′-exonucleases, mostly found in a complex called the exosome. These pathways and

Post-transcriptional cis-elements

In most cases, stage-specific post-transcriptional regulation is mediated through the 3′UTRs of mRNAs [14]. In few cases, specific regions within 3′UTRs have been delineated as cis-elements in these organisms mainly by deletion analysis in reporter systems. Table 1 provides an overview on most currently known cis-elements and their mechanism of action. The availability of genome sequences, and data from microarray studies have already facilitated the use of bioinformatics to identify cis

Trans-regulators

Consistent with the predominance of post-transcriptional mechanisms, analysis of TriTryp genomes has revealed a large number of conserved putative RNA binding proteins (RBPs) [35, 36, 37••]. Despite serious attempts to affinity purify proteins that bind known cis-elements [14] and to evaluate candidate factors [36], only a few experimentally verified regulatory RBPs, including the U-rich binding proteins UBP1 and UBP2 of T. cruzi involved in the regulation of stage-specific small mucin mRNAs [

Post-translational regulation

Very little is known about post-translational gene regulation in these parasites. Their genomes harbor relatively high number of putative kinase genes [46]. The phosphorylation status of Crithidia's RBPs is correlated with their cell cycle phase dependent effect on mRNA stability [38]. Molecular variants of many proteins that are potentially generated via post-translational modifications have been identified in protein 2D gels [4•, 47]. Protein degradation partly accounts for stage-specific

Conclusions and future challenges

The exciting discovery of SIDERs and their role in the regulation of gene expression suggests that Leishmania, unlike its trypanosomatid cousins, has opted to combat the invasion of retroposons by rendering them inactive, and subsequently propagated and conscripted these elements to promote its own agenda-the large-scale regulation of its own gene expression. This might have allowed specific adaptations of the parasite to its unique niches (e.g. phagolysosomes). The molecular dissection of

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We apologize to colleagues whose work has not been mentioned owing to space limitations. We thank members of the lab for their contributions to our work in this domain. SH is supported by a Canadian Institute of Health Research (CIHR) fellowship. BP is a member of a CIHR group on host-pathogen interactions. This work was supported by funds from CIHR (MOP-12182).

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