Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei
Introduction
Salivarian trypanosomes are protists which infect mammals in sub-Saharan Africa. The parasites multiply in the blood and tissue fluids of their mammalian hosts, and in the midgut of the insect vector, the Tsetse fly. In trypanosomes and other members of the order Kinetoplastida, the first seven to nine enzymes of glycolysis, and various other enzymes, are compartmentalised in a microbody called the glycosome which is related to the peroxisomes of mammals and yeasts [1]. In bloodstream trypanosomes, glycolysis is the major source of ATP, and most phosphoglycerate kinase activity is found within the glycosome, where it is responsible for regenerating the ATP which is consumed by the hexokinase and phosphofructokinase reactions [2]. In contrast, in the procyclic form, which grows in Tsetse, mitochondrial metabolism is more important than glycolysis [3]. In procyclics, most phosphoglycerate kinase is found in the cytosol. Cytosolic phosphoglycerate kinase (PGKB) is encoded by the PGKB gene, which is located directly upstream of the gene-encoding glycosomal phosphoglycerate kinase, PGKC, on Trypanosoma brucei chromosome I [4]. The developmentally regulated expression of the two PGKs is essential for trypanosome survival, as expression of cytosolic PGK activity in bloodstream trypanosomes inhibits their growth [5].
Most Kinetoplastid genes are transcribed as long polycistronic precursors, which are subsequently cleaved to form monocistronic mRNAs by a trans splicing reaction in which a 39 nt-capped leader sequence is added to the 5′-end of each mRNA, and by polyadenylation. There is as yet no evidence for any regulation of transcription by RNA polymerase II during trypanosome growth. The polycistronic mode of transcription of the PGK genes was demonstrated nearly 20 years ago by Gibson et al. [6], who documented the existence of precursor RNAs spanning the gap between PGKB polyadenylation site and the PGKC trans splicing acceptor site [6]. PGKC mRNA is at least 20-fold more abundant in bloodstream forms than in procyclic forms, while PGKB mRNA is at least 10-fold regulated [7], [8]. By transient transfection of plasmids which encoded chloramphenicol acetyltransferase (CAT), we demonstrated that the 3′-untranslated regions (UTRs) of the PGKB and PGKC mRNAs were sufficient to cause procyclic- and bloodstream-form-specific expression, respectively [7]. A CAT reporter mRNA with a PGKB 3′-UTR was very unstable (half-life, 5–10 min) in bloodstream trypanosomes, and the region causing instability was mapped to a poly(U) tract [8].
To analyse regulation of PGKB mRNA degradation further, we assessed the effects of depletion of enzymes involved in mRNA degradation in bloodstream forms. A complex of 3′→5′ exonucleases, the exosome, was shown to be limiting in the initiation of degradation of the CAT reporter RNA with a PGKB 3′-UTR [9], but there were no major effects on the abundance of the reporter mRNA, or on the abundances of native PGKB and PGKC mRNAs. In contrast, depletion of the 5′→3′ exonuclease homologue XRNA was found to cause deregulation of both PGKB and PGKC mRNA: PGKC mRNA became detectable in procyclic forms, and PGKB mRNA levels increased in bloodstream forms [10].
In this paper, we concentrate on the mechanism of regulation of the bloodstream-specific PGKC mRNA.
Section snippets
Trypanosome culture and transfection
Bloodstream and procyclic form T. brucei were cultured and transfected as previously described [11], [12]. The strains used, which constitutively express bacteriophage T7 polymerase and the Tn10 Tet-repressor, are strain Lister 427 containing pHD514 (T7 polymerase, G418 resistance) and pHD1313 (tet repressor, phleomycin resistance) [13].
To obtain cells expressing CAT mRNAs, expression plasmids were linearised at a NotI-site within a segment of the plasmid from either an rRNA spacer region or
PGKC mRNA degradation in bloodstream and procyclic trypanosomes
We had previously shown that PGKC and PGKB regulation was disrupted by depletion of the 5′→3′ exonuclease XRNA [10]. To analyse the mechanism of this effect, we treated bloodstream and procyclic trypanosomes with Sinefungin [19], [20], which inhibits 5′-capping of the spliced leader RNA and therefore indirectly inhibits trans splicing [21], [22], [23]. Results are shown in Fig. 1. Thirty minutes after Sinefungin addition, two bands migrating at about 7 and 11 kb became visible (Fig. 1A and B).
Discussion
The main aim of the work described in this paper was the identification of the 3′-UTR sequence responsible for the down-regulation of PGKC enzyme and PGKC mRNA in procyclic trypanosomes. To map the sequences, we used a reporter plasmid expressing CAT mRNAs with the PGKC 3′-UTR, transcribed by either T7 polymerase or RNA polymerase II. Procyclic trypanosomes transfected with plasmids containing the complete PGKC 3′-UTR sequence expressed 100-fold less CAT activity than cells transfected with a
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft, and by a DAAD stipend to AR and a Croucher Foundation stipend to CHL. Experimental work was done by Claudia Colasante (Fig. 3, Fig. 4, supervised by Lys Guilbride and Frank Voncken), Ana Robles (Fig. 5, Fig. 6A), Chi-Ho Li (Fig. 1, Fig. 6C) and Angela Schwede (Fig. 6B). Corinna Benz and Lys Guilbride contributed to plasmid construction and design (Fig. 3). Christine Clayton wrote the DFG grant applications and the paper. We thank
References (39)
- et al.
Metabolic aspects of glycosomes in trypanosomatidae—new data and views
Parasitol Today
(2000) - et al.
New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle
J Biol Chem
(2005) - et al.
Post-transcriptional control of the differential expression of phosphoglycerate kinase genes in Trypanosoma brucei
J Mol Biol
(1988) - et al.
The 3′-untranslated regions from the Trypanosoma brucei phosphoglycerate kinase genes mediate developmental regulation
Gene
(1995) - et al.
Vectors for inducible over-expression of potentially toxic gene products in bloodstream and procyclic Trypanosoma brucei
Mol Biochem Parasitol
(1997) - et al.
Messenger RNA processing sites in Trypanosoma brucei
Mol Biochem Parasitol
(2005) - et al.
Effect of multiple downstream splice sites on polyadenylation in Trypanosoma brucei
Mol Biochem Parasitol
(1998) - et al.
Cytoplasmic degradation of splice-defective pre-mRNAs and intermediates
Mol Cell
(2003) - et al.
Leishmania chagasi: the alpha-tubulin intercoding region results in constant levels of mRNA abundance despite protein synthesis inhibition and growth state
Exp Parasitol
(2005) - et al.
Developmental regulation of heat shock protein 83 in Leishmania
J Biol Chem
(2001)
Amastin mRNA abundance in Trypanosoma cruzi is controlled by a 3′-untranslated region position-dependent cis-element and an untranslated region-binding protein
J Biol Chem
RNAi interference of XPO1 and Sm genes and their effect on the spliced leader RNA in Trypanosoma brucei
Mol Biochem Parasitol
Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei
Proteomics
The DNA sequence of chromosome I of an African trypanosome: gene content, chromosome organisation, recombination and polymorphism
Nucleic Acids Res
The roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei
Biochem J
Expression of the human RNA-binding protein HuR in Trypanosoma brucei induces differentiation-related changes in the abundance of developmentally-regulated mRNAs
Nucleic Acids Res
A role for the exosome in the initiation of degradation of unstable mRNAs
RNA
The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context
Nucleic Acids Res
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Current address: Department of Biological Sciences, Hardy Building, Room 143, Cottingham Road, University of Hull, Hull HU6 7RX, United Kingdom.
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Current address: Division of Cellular Immunology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.