Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei

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Abstract

In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3′-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3′→5′ exonuclease complex) or the 5′→3′ exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes.

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

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    1

    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.

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