The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes

Abstract

In trypanosomes, the predominant mechanisms of regulation of gene expression are post-transcriptional. The DEAD-box RNA helicase DHH1 was identified in a screen for gene products that are necessary for the instability of the GPI-PLC mRNA in insect-stage trypanosomes. Expression of an ATPase-deficient dhh1 mutant caused a rapid growth arrest associated with a decrease in polysomes, an increase in P-bodies and a slight decrease in average mRNA levels. However, the effect of dhh1 mutant expression on both turnover and translational repression of mRNAs was selective. Whereas there was little effect on the stability of constitutive mRNAs, the control of a large cohort of developmentally regulated mRNAs was reversed; many mRNAs normally downregulated in insect-stage trypanosomes were stabilized and many mRNAs normally upregulated decreased in level. One stabilised mRNA, ISG75, was characterised further. Despite the overall decrease in polysomes, the proportion of the ISG75 mRNA in polysomes was unchanged and the result was ISG75 protein accumulation. Our data show that specific mRNAs can escape DHH1-mediated translational repression. In trypanosomes, DHH1 has a selective role in determining the levels of developmentally regulated mRNAs.

Fig. 1.

RNAi knockdown of DHH1 stabilises the GPI-PLC mRNA in procyclic cells. (A) GPI-PLC mRNA, (B) cell proliferation and (C) DHH1 protein were followed over a time course after induction of RNAi. For DHH1 protein, quantification was done by comparison with a titration of cell equivalents of the parental cell line T. brucei Lister 427 pLEW29:pLEW13 (Wirtz et al., 1999). One representative experiment of three is shown.

Fig. 2.

Growth phenotype after expression of DHH1 wild type and mutants. Cell proliferation after induction of DHH1, dhh1 E182Q, dhh1 R74A/K76A and dhh1 G332 transgenes. The increase in expression of DHH1 was determined by quantitative western blotting.

Fig. 3.

Subcellular localisation of DHH1 wild type and mutants. (A) Images of representative cells expressing eYFP-DHH1 wild type and mutants after 12 hours induction and the effect of heat shock and puromycin on the subcellular distribution. The percentage of cells (n=150) with microscopically visible P-bodies in each case is shown below. (B) Effect of expression of DHH1 wild type and mutants on subcellular localisation of SCD6-eYFP, an independent P-body marker. Representative images are shown 0 and 24 hours after induction. Quantitation of P-body size is shown below; 30 cells per time point were measured and P-values of a Student's t-test comparing uninduced and induced cells are shown.

Fig. 4.

Changes in total mRNA and polysomes after dhh1-E182Q induction. (A) Total mRNA was measured after induction of dhh1 E182Q by probing northern blots for the spliced leader present on all mRNAs. Values were normalised using the 0-hour time point. (B) Polysome analysis after induction of dhh1-E182Q expression. (C) Cell proliferation after induction of dhh1 E182Q to determine the point of arrest.

Fig. 5.

Changes in mRNA after induction of expression of dhh1-E182Q or DHH1 transgenes. (A) Microarray dataset shown as a plot of the ratio of average intensities for each spot before and after dhh1-E182Q induction (the vast majority of genes are represented by a single spot). The most upregulated and most downregulated mRNAs (filtered as described in the text, see Tables 1 and 2) are coloured in red and blue, respectively. (B) The expression of four upregulated and two downregulated mRNAs, marked by black squares in A, was measured over a time course after induction of expression of dhh1 E182Q. Data from two independent experiments are shown. RNA from the parental cell line (Lister 427 PTT) with and without tetracycline is shown as a control. The data were normalised using the 0-hour time point. (C) Microarray dataset shown as plot of the ratio of average intensities for each spot before and after DHH1 transgene induction. For comparison, the mRNAs most up- and downregulated by the expression of dhh1 E182Q are coloured in red and blue, respectively.

Fig. 6.

Developmental regulation of mRNAs identified in the dhh1-E182Q microarray experiment. Quantification of relative mRNA expression in bloodstream and procyclic forms using northern blots with RNA from procyclic (‘P’) and bloodstream form cells (‘B’). Note that Tb927.5.2170, Tb927.5.2230 and Tb927.5.2260 are 99% identical and are recognised by the same probe. Data of two independent experiments are shown (one representative blot and average value of the quantification).

Fig. 7.

Increase in mRNA stability following expression of dhh1 E182Q. RNA half-lives were estimated by quantification over a time course after actinomycin-D addition using cells before or 12 hours after dhh1-E182Q induction. Average data of three (ISG75) or two (Tb11.01.3915 and Tb11.47.0021) independent experiments are shown. Standard deviations are indicated as error bars (ISG75).

Fig. 8.

ISG75 protein and mRNA after dhh1-E182Q induction. (A) ISG75 protein expression was determined by western blotting over a time course after dhh1-E182Q induction. A titration of bloodstream-form (BSF) cell equivalents was used to estimate expression levels. BiP expression is shown as a loading control (Bangs et al., 1993). (B) Distribution of ISG75 mRNA on a polysome analysis before and 12 hours after dhh1-E182Q induction analysed by northern blotting. The distributions shown are relative with the total set at 100%. eIF4E3 mRNA was used as a control.

Fig. 9.

Comparison of the dhh1-E182Q and the BSF:PCF microarray. Effect of dhh1-E182Q expression on (A) mRNAs developmentally upregulated in bloodstream forms (B:P >2.5), and (C) mRNAs developmentally downregulated in bloodstream forms (B:P <0.4). In both cases the B:P ratio is on the y-axis and the dhh1-E182Q-induced:uninduced value is on the x-axis. In A, the group of strongly developmentally regulated mRNAs unaffected by dhh1 E182Q are circled and two genes of this group have been analysed by northern blot (B).