Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.

Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

An RNAi screen of the RRM-domain proteins of Trypanosoma brucei.

In eukaryotes, proteins containing RNA Recognition Motifs (RRMs) are involved in many different RNA processing reactions, RNA transport, and mRNA decay. Kinetoplastids rely extensively on post-transcriptional mechanisms to control gene expression, so RRM domain proteins are expected to play a prominent role. We here describe the results of an RNA interference screen targeting 37 of the 72 RRM-domain proteins of Trypanosoma brucei. RNAi targeting 8 of the genes caused clear growth inhibition in bloodstream trypanosomes, and milder effects were seen for 9 more genes. The small, single-RRM protein TbRBP3 specifically associated with 10 mRNAs in trypanosome lysates, but RBP3 depletion did not affect the transcriptome.

Small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of F-box protein mRNAs in bloodstream trypanosomes.

In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3'-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, TbUBP1 and TbUBP2, containing a single RNA recognition motif, in trypanosome gene expression. TbUBP1 and TbUBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of TbUBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3'-untranslated region and involved stabilization of the mRNA. Depletion of TbUBP2 and TbUBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to TbUBP2 or TbUBP1, with some preference for CFB1. We suggest that TbUBP1 and TbUBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.

Troglitazone induces differentiation in Trypanosoma brucei.

Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor gamma. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.

Functional analysis of Trypanosoma brucei PUF1.

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.

The transcriptomes of Trypanosoma brucei Lister 427 and TREU927 bloodstream and procyclic trypomastigotes.

We describe developmentally regulated genes in two strains of Trypanosoma brucei: the monomorphic strain Lister 427 and the pleomorphic strain TREU927. Expression patterns were obtained using an array of 24,567 genomic fragments. Probes were prepared from bloodstream-form or procyclic-form trypanosomes. Fourteen procyclic-specific and 77 bloodstream-specific signals were obtained from sequences matching variant surface glycoprotein or associated genes, and a further 17 regulated sequences were repetitive or transposable-element-related. Two hundred and eighty-six regulated spots corresponded to mRNAs from other protein-coding genes; these spots represent 191 different proteins. Regulation of 113 different genes (79 from procyclic forms, 34 from bloodstream-forms) was supported by at least two independent experiments or criteria; of these, about 60 were novel. Only two genes -- encoding HSP83 and an importin-related protein -- appeared to be regulated in the TREU927 strain only. Our results confirmed previous estimates that 2% of trypanosome genes show developmental regulation at the mRNA level.

The renal retinoid system: time-dependent activation in experimental glomerulonephritis.

Retinoids reduce renal damage in rat experimental glomerulonephritis. It is unknown, however, how local and systemic retinoid pathways respond to renal injury. We used a rat model of artificially induced acute anti-Thy1.1-nephritis (THY-GN). We examined the extrarenal and glomerular expression of the retinol (RoDH) and retinal (RalDH) dehydrogenases 1 and 2 as well as the expression of the retinoic acid (RAR) and retinoid X (RXR) receptor subtypes alpha, beta, and gamma. Furthermore, we investigated serum and glomerular retinoid concentration patterns. On days 3, 7, and 14, we compared nonnephritic rats (control group; CON) to THY-GN rats with respect to systolic blood pressure and glomerular cell count per cross section. Systolic blood pressure and glomerular cell count were significantly higher in THY-GN rats on days 7 and 14 (P < 0.001). We found a 60% reduction in expression levels for retinoid receptors and dehydrogenases in nephritic glomeruli on day 3, but a threefold increase on day 7 (P < 0.001 vs. CON). The same applies to RAR alpha protein. Hepatic expression of retinoid receptors was not influenced. On day 14, glomerular expression levels for retinoid receptors and retinoid-metabolizing enzymes had returned to a normal level, glomerular cell count being still increased. Administering 13-cis retinoic acid (isotretinoin) lowered blood pressure and glomerular cell count in nephritic rats but failed to influence the glomerular expression of retinoid receptors or retinoid-metabolizing enzymes. Our data document a stimulation of glomerular retinoid-synthesizing enzymes and expression of retinoid receptors in the early repair phase of THY-GN, suggesting activation of this system in acute renal disease.