Functional analysis of Trypanosoma brucei PUF1

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Abstract

The genomes of Trypanosoma brucei, Leishmania major and Trypanosoma cruzi each encode 10 proteins with PUF domains. PUF domain proteins from yeast and metazoa have been shown to bind RNA and to regulate mRNA stability and translation. Phylogenetic analysis suggested that the PUF proteins were duplicated and diverged early in evolution, and that most PUF proteins were lost during the evolution of mammals. Depletion of any of the first nine T. brucei PUF protein mRNAs by RNA interference had no effect on cell growth; combined depletion of PUF1 and PUF3, PUF3 and PUF4, and PUF1 and PUF4 mRNAs also had no effect. In conflict with a previous report, procyclic trypanosomes lacking PUF1 genes grew normally and we could find no evidence that PUF1 is required for growth of trypanosomes in culture. Depletion or elimination of PUF1 mRNA did not affect the abundances of any other mRNAs, as detected in microarray analysis, and also had minimal effects on the proteome. (In control experiments, treatment of bloodstream and procyclic cells with 100 ng/ml tetracycline also had no detectable effects on the transcriptome and proteome.) PUF1 preferentially bound to retroposon RNAs and was not associated with polysomes. We suggest that, as in yeast, there may be functional redundancy among the Kinetoplastid PUF proteins, or they may be involved in fine-tuning gene expression together with other proteins. Alternatively, PUF proteins may be needed in differentiating trypanosomes or in non-culturable life-cycle stages.

Introduction

Proteins of the PUF family have been found in virtually all eukaryotes examined [1]. The two founding members of this family were Drosophila Pumilio and C. elegans FBF. In Drosophila, Pumilio protein is known to bind the 3′-untranslated region (3′-UTR) of hunchback mRNA, leading to an increase in the rate of deadenylation and repression of translation of this mRNA [2], [3], while in C. elegans, FBF protein binds the 3′-UTR of fem-3 mRNA and leads to repression of translation [4]. PUF proteins in yeast and slime moulds also repress expression of target mRNAs by binding to sequences in the 3′-UTR [5], [6], [7]. PUF proteins are characterized by the presence of eight consecutive repeats (Puf repeats), each consisting of approximately 40 amino acids. Each repeat contains 3 alpha helices, and the 8 repeats forms an extended crescent [8], [9]. The Puf repeat region is necessary and sufficient to bind to specific RNA sequences and to effect some of the protein's biological functions. The inner surface of the crescent carries the conserved aromatic and charged amino acid residues that are likely to bind RNA, and the outer surface can contact other proteins.

The kinetoplastid genome sequences predict the presence of 10 PUF protein family members [10]. More primitive organisms, including C. elegans and S. cerevisiae, possess multiple PUF proteins, whereas only one PUF protein (with two isoforms) is present in Drosophila. Vertebrates also have a smaller number of PUF proteins [1], [4], [11]. Thus, it would appear that multiple Puf-family proteins may have emerged early in evolution, but many were lost during evolution of arthropods and vertebrates.

Functional studies of protist Puf-protein family members have so far mainly been limited to in vitro RNA binding analyses and so far no in vivo regulatory targets have been definitively identified. T. cruzi PUF6 [12] and PUF1 [10], and the two Puf proteins of P. falciparum, PfPuf1 and PfPuf2 [13], were shown to bind specifically to the Drosophila hunchback Puf recognition sequence in vitro but no further functional information on these proteins is available. T. cruzi PUF3, 5 and 8 did not show binding to the sequence in a three-hybrid assay [10].

The most extensive functional studies of protist PUF proteins so far have been of TbPUF1, which was found in a two-hybrid screen for proteins which interact with TbESAG8, a leucine repeat protein [14]. PUF1 mRNA was shown to be expressed at equal levels in both insect and bloodstream-form parasites. Attempts to disrupt the two PUF1 alleles in both bloodstream-form and procyclic-form trypanosomes by classical homologous recombination failed, but a conditional PUF1 mutant cell line in bloodstream forms showed only a very slight growth defect upon PUF1 down-regulation. Cells that over-expressed an epitope-tagged version of PUF1 had a significant growth defect and a reduced infectivity in mice.

TbESAG8 genes are located near telomeres and in sub-telomeric regions [15]. They are transcribed as part of the telomeric RNA polymerase I transcription units called “VSG expression sites”, which contain up to 10 “expression site associated genes” (ESAGs) and terminate with a gene encoding the variant surface glycoprotein (VSG). The results of messenger mRNA stability assays suggested that TbPUF1 might regulate stability of specific expression site (ES) derived mRNAs (e.g. ESAG8 and VSG221) in trypanosomes [14]. The interaction between TbPUF1 and ESAG8 was confirmed in pull-down assays, but the proportions of ESAG8 and PUF1 which were interacting were not measured and the biological significance of the interaction was unclear since TbPUF1 was found in the cytoplasm [14], whereas TbESAG8 is localized to the nucleolus [16].

In this report we investigated the possible function of TbPUF1 in more detail, and extended the study to all other T. brucei PUF proteins.

Section snippets

Trypanosome culture and transfection

Trypanosomes expressing the Tet repressor [17], [18] were cultured and transfected as previously described [17], [19], [20]. To induce tetracycline-regulatable promoters, tetracycline was added to 100 ng ml−1. RNA and protein were prepared only from exponentially growing cells (less than 2 × 106 ml−1 for bloodstream forms and 5 × 106 ml−1 for procyclic forms). For the knockout, procyclic form 427 cells were sequentially transfected with SacI/ApaI-linearised pHD1639 (PUF1-BSD) and pHD1641 (PUF1-NPT)

The 10 PUF proteins of T. brucei

There are 10 T. brucei PUF proteins whose structures resemble those of the T. cruzi PUFs [10]. We did however note the presence of a predicted transmembrane helix in TbPUF3 and a putative mitochondrial localization signal in TbPUF5. Since the relationship of kinetoplastid PUF proteins to those of other eukaryotes had not previously been investigated, we generated a phylogenetic tree using only the PUF domains from 90 Puf family members (Fig. 1). We found that PUF proteins of vertebrates

Discussion

In Drosophila, mammalian cells and yeast, PUF proteins regulate mRNA stability and translation. The aim of this study was to define genuine in vivo targets for regulation by PUF1 in T. brucei. We applied two criteria: first, the target mRNA is expected to bind to PUF1 in vivo; and second, alterations in PUF1 expression should result in a change in the level of the mRNA or the encoded protein. We used bloodstream forms in which PUF1 expression had been down-regulated by RNAi, procyclic cells

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinshaft, the German-Israeli foundation (Germany) and the Medical Research Council (UK). We thank George Cross for initially sending DNA clones, and Laurie Read for the anti-PABP antibody. All of the experimental work was done by V.-D. Luu, who also wrote a large proportion of the paper. S. Brems (supervised by J. Hoheisel) made the microarrays and assisted with some of the microarray experiments, D.L. Guilbride supervised the initial phase

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    • Cited by (0)

    1

    Current address: Division of Molecular Genome Anlaysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany.

    2

    Current address: Division of Cellular Immunology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

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