Contributions of the procyclin 3′ untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei

Abstract

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3′ untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a ∼6-fold increase in the level of steady state mRNA and >30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3′ UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3′ UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Introduction

In the course of a digenetic life cycle, Trypanosoma brucei undergoes a series of morphological and physiological changes which allow it to adapt to extensive changes in environment. Differentiation from one life cycle stage to the next requires a carefully regulated programme of gene expression. In some cases stage-specific genes are coordinately transcribed from a single polycistronic array, but still require further fine-tuning of expression by a range of post-transcriptional mechanisms 1, 2, 3, 4, 5, 6.

All life cycle stages of T. brucei are covered by a dense glycoprotein coat. Bloodstream and metacyclic forms, which are exposed to the mammalian immune system, are covered by a coat of variant surface glycoprotein (VSG). The VSG has the dual function of preventing complement-activated lysis by the alternative pathway and allowing the parasite to escape immune recognition by periodic switching to a new antigen type [7]. In contrast, the procyclin (or PARP) coat of the procyclic and epimastigote forms in the tsetse fly [8]does not undergo antigenic variation, is potentially highly immunogenic and does not protect against lysis. Premature expression of procyclin or loss of the VSG coat could therefore make the parasite vulnerable on two fronts.

When bloodstream forms are triggered to differentiate, the change of coat occurs extremely rapidly, with the expression of procyclin preceding the loss of VSG 9, 10, 11. In pleomorphic populations with a large proportion of short stumpy forms that are able to differentiate synchronously, >90% of the parasites express procyclin within 2 h and lose VSG after 4 h [11]. The exchange of coat occurs more slowly in monomorphic forms [9]which first need to reach a particular point in the cell cycle before they are competent to differentiate [11].

Procyclins are subject to a series of controls that prevent their untimely expression in the mammalian host 1, 3, 12. In bloodstream forms transcription initiation occurs at ≈5–15% of the rate in procyclic forms 13, 14. Despite this, little or no mature mRNA and no protein can be detected [9]. By tagging individual procyclin expression sites with unique genetic markers, it could be demonstrated that transcription is confined to the beginning of the procyclin expression sites in bloodstream forms, and that an increase in both transcription initiation and elongation occurs as the parasites differentiate into procyclic forms [14]. This effect is independent of the sequence since it was observed with an endogenous gene (GRESAG 2; [15]) and also with integrated reporter genes. The same factors which act in concert to trigger differentiation—a drop in temperature, the addition of citrate and cis-aconitate, and the appropriate medium—stimulate elongation of transcription from the procyclin expression sites, both singly and in combination, and at the same time reduce transcription driven by the VSG and ribosomal promoters [14]. It has recently been shown that a 40 kDa polypeptide is able to bind to the coding strands of all three promoters and to a lesser extent to the non-coding strand of the procyclin promoter [16]. This raises the possibility that the reciprocal activities of the VSG and procyclin expression sites may be a consequence of rerouting transcription factors at different stages of the life cycle.

There is evidence that labile proteins block procyclin expression in bloodstream forms. When treated with inhibitors of protein synthesis, these forms accumulate procyclin mRNA 17, 18. This effect is not due to a stimulation of transcription [18]. The transcripts are polyadenylated and exported from the nucleus, raising the possibility that either an endonuclease or a specific inhibitor of procyclin RNA processing has been depleted from the cells. The procyclin 3′ untranslated region (UTR) also plays a role in regulating expression. We have recently identified three elements in the 3′ UTR which have a major influence on expression in procyclic forms 4, 5. There are two positive elements which offset the effect of a central negative element that destabilises the mRNA and inhibits translation. One of the positive elements is a conserved 16-mer [4]which also occurs in the 3′ UTR of GARP (glutamic acid/alanine-rich protein), the procyclin analogue of T. congolense [19]. The second positive element is destroyed by the deletion of the first 40 bp of the procyclin 3′ UTR [5].

It has recently been shown that regulatory sequences in the VSG mRNA may have different effects depending on the stage of the life cycle. An element extending from the end of the coding region to the polyadenylation site up-regulates expression 2–3-fold in bloodstream forms, when compared with a control construct lacking trypanosome sequences downstream of the reporter gene, and down-regulates it to a similar extent in procyclic forms [6]. The increased expression in bloodstream forms correlated with an increase in RNA stability, but the converse was not true for procyclic forms. In the same study it was shown that a portion of the procyclin 3′ UTR had a negative effect on expression in bloodstream forms (2.5-fold less than the control), but a positive effect of a similar magnitude in procyclic forms, again suggesting that a single element might act as a stage-specific switch.

We have investigated the role of the procyclin 3′ UTR on stage-specific expression by using a panel of deletion mutants. Our results indicate that individual elements tend to have the same effect in both bloodstream and procyclic forms, but to different extents. This can best be explained by a model in which there is no absolute regulation, but rather a change in equilibrium between two forms of procyclin mRNA; a stable, efficiently translated form which predominates in procyclic trypanosomes and a labile form which predominates in the bloodstream.