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The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages.

Vembar SS, Macpherson CR, Sismeiro O, Coppée JY, Scherf A - Genome Biol. (2015)

Bottom Line: Using deep sequencing of affinity-purified PfAlba1-associated RNAs, we identify 1193 transcripts that directly bind to PfAlba1 in trophozoites.For 105 such transcripts, 43 % of which are uncharacterized and 13 % of which encode erythrocyte invasion components, the steady state levels significantly change at this stage, evidencing a role for PfAlba1 in maintaining mRNA homeostasis.This mode of post-transcriptional regulation may be especially important for P. falciparum erythrocyte invasion components that have to be assembled into apical secretory organelles in a highly time-dependent manner towards the end of the parasite's asexual lifecycle.

View Article: PubMed Central - PubMed

Affiliation: Unité Biologie des Interactions Hôte-Parasite, Département de Parasites et Insectes Vecteurs, Institut Pasteur, Paris, 75015, France. shruthi-sridhar.vembar@pasteur.fr.

ABSTRACT

Background: Transcriptome-wide ribosome occupancy studies have suggested that during the intra-erythrocytic lifecycle of Plasmodium falciparum, select mRNAs are post-transcriptionally regulated. A subset of these encodes parasite virulence factors required for invading host erythrocytes, and are currently being developed as vaccine candidates. However, the molecular mechanisms that govern post-transcriptional regulation are currently unknown.

Results: We explore the previously identified DNA/RNA-binding protein PfAlba1, which localizes to multiple foci in the cytoplasm of P. falciparum trophozoites. We establish that PfAlba1 is essential for asexual proliferation, and subsequently investigate parasites overexpressing epitope-tagged PfAlba1 to identify its RNA targets and effects on mRNA homeostasis and translational regulation. Using deep sequencing of affinity-purified PfAlba1-associated RNAs, we identify 1193 transcripts that directly bind to PfAlba1 in trophozoites. For 105 such transcripts, 43 % of which are uncharacterized and 13 % of which encode erythrocyte invasion components, the steady state levels significantly change at this stage, evidencing a role for PfAlba1 in maintaining mRNA homeostasis. Additionally, we discover that binding of PfAlba1 to four erythrocyte invasion mRNAs, Rap1, RhopH3, CDPK1, and AMA1, is linked to translation repression in trophozoites whereas release of these mRNAs from a PfAlba1 complex in mature stages correlates with protein synthesis.

Conclusions: We show that PfAlba1 binds to a sub-population of asexual stage mRNAs and fine-tunes the timing of translation. This mode of post-transcriptional regulation may be especially important for P. falciparum erythrocyte invasion components that have to be assembled into apical secretory organelles in a highly time-dependent manner towards the end of the parasite's asexual lifecycle.

No MeSH data available.


Related in: MedlinePlus

PfAlba1 overexpression reduces intra-erythrocytic growth of P. falciparum. a Protein lysates prepared from empty vector or PfAlba1-Ty1 transfectants grown in the presence of increasing concentrations of blasticidin-S (BS; 2.5–20 μg/ml) were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies, anti-PfAlba1 antibodies, or anti-PfAlba4 antibodies. PfAldolase served as a loading control. b The levels of PfAlba1-Ty1, as detected by the anti-Ty1 antibodies in (a), were measured by densitometry. For each BS concentration, after normalizing the PfAlba1-Ty1 signal to the PfAldolase signal, data were normalized to the 2.5 μg/ml sample. Data represent the means of a minimum of three independent experiments ± standard error (S.E.; error bars). c The growth of empty vector (left panel) or PfAlba1-Ty1 (right panel) parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/ml). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± S.E. (error bars). d The ~48 h IDC of empty vector or PfAlba1-Ty1 transfectants was monitored by flow cytometry. The y-axis denotes the percentage of ring stages at each time point. The vertical dashed line represents one replication cycle. Data represent the means of three independent experiments ± S.E. (error bars)
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Fig2: PfAlba1 overexpression reduces intra-erythrocytic growth of P. falciparum. a Protein lysates prepared from empty vector or PfAlba1-Ty1 transfectants grown in the presence of increasing concentrations of blasticidin-S (BS; 2.5–20 μg/ml) were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies, anti-PfAlba1 antibodies, or anti-PfAlba4 antibodies. PfAldolase served as a loading control. b The levels of PfAlba1-Ty1, as detected by the anti-Ty1 antibodies in (a), were measured by densitometry. For each BS concentration, after normalizing the PfAlba1-Ty1 signal to the PfAldolase signal, data were normalized to the 2.5 μg/ml sample. Data represent the means of a minimum of three independent experiments ± standard error (S.E.; error bars). c The growth of empty vector (left panel) or PfAlba1-Ty1 (right panel) parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/ml). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± S.E. (error bars). d The ~48 h IDC of empty vector or PfAlba1-Ty1 transfectants was monitored by flow cytometry. The y-axis denotes the percentage of ring stages at each time point. The vertical dashed line represents one replication cycle. Data represent the means of three independent experiments ± S.E. (error bars)

Mentions: Before functionally characterizing PfAlba1-Ty1, we explored if we could modulate the expression levels of the tagged protein during the P. falciparum IDC, and if so, whether the parasite had a tolerance threshold for PfAlba1-Ty1 levels. When we treated 3D7+PfAlba1-Ty1 parasites that were initially growing with 2.5 μg/ml blasticidin-S (BS) in the growth medium with increasing amounts of BS for two days, PfAlba1-Ty1 protein levels gradually increased (Fig. 2a), reaching a maximum of fivefold upregulation at 20 μg/ml BS relative to 2.5 μg/ml BS (Fig. 2b). We also observed that the levels of endogenous PfAlba1 increased in a BS dose-dependent manner (Fig. 2a; Figure S2a in Additional file 1), but not of a second Alba family protein PfAlba4 (Fig. 2a) and two other orthologs, PfAlba2 and PfAlba3 (data not shown), indicating that overexpression of PfAlba1-Ty1 directly affected endogenous PfAlba1 levels. Note that, even at 2.5 μg/ml BS, PfAlba1-Ty1 transfectants express higher levels of total PfAlba1 compared with the empty vector control (Figs. 1b and 2a).Fig. 2


The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages.

Vembar SS, Macpherson CR, Sismeiro O, Coppée JY, Scherf A - Genome Biol. (2015)

PfAlba1 overexpression reduces intra-erythrocytic growth of P. falciparum. a Protein lysates prepared from empty vector or PfAlba1-Ty1 transfectants grown in the presence of increasing concentrations of blasticidin-S (BS; 2.5–20 μg/ml) were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies, anti-PfAlba1 antibodies, or anti-PfAlba4 antibodies. PfAldolase served as a loading control. b The levels of PfAlba1-Ty1, as detected by the anti-Ty1 antibodies in (a), were measured by densitometry. For each BS concentration, after normalizing the PfAlba1-Ty1 signal to the PfAldolase signal, data were normalized to the 2.5 μg/ml sample. Data represent the means of a minimum of three independent experiments ± standard error (S.E.; error bars). c The growth of empty vector (left panel) or PfAlba1-Ty1 (right panel) parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/ml). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± S.E. (error bars). d The ~48 h IDC of empty vector or PfAlba1-Ty1 transfectants was monitored by flow cytometry. The y-axis denotes the percentage of ring stages at each time point. The vertical dashed line represents one replication cycle. Data represent the means of three independent experiments ± S.E. (error bars)
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Related In: Results  -  Collection

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Fig2: PfAlba1 overexpression reduces intra-erythrocytic growth of P. falciparum. a Protein lysates prepared from empty vector or PfAlba1-Ty1 transfectants grown in the presence of increasing concentrations of blasticidin-S (BS; 2.5–20 μg/ml) were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies, anti-PfAlba1 antibodies, or anti-PfAlba4 antibodies. PfAldolase served as a loading control. b The levels of PfAlba1-Ty1, as detected by the anti-Ty1 antibodies in (a), were measured by densitometry. For each BS concentration, after normalizing the PfAlba1-Ty1 signal to the PfAldolase signal, data were normalized to the 2.5 μg/ml sample. Data represent the means of a minimum of three independent experiments ± standard error (S.E.; error bars). c The growth of empty vector (left panel) or PfAlba1-Ty1 (right panel) parasites was measured by flow cytometry for 5 days in the presence of the indicated concentrations of BS (in μg/ml). The y-axis denotes the percentage parasitemia at each time point. Data represent the means of a minimum of three independent experiments ± S.E. (error bars). d The ~48 h IDC of empty vector or PfAlba1-Ty1 transfectants was monitored by flow cytometry. The y-axis denotes the percentage of ring stages at each time point. The vertical dashed line represents one replication cycle. Data represent the means of three independent experiments ± S.E. (error bars)
Mentions: Before functionally characterizing PfAlba1-Ty1, we explored if we could modulate the expression levels of the tagged protein during the P. falciparum IDC, and if so, whether the parasite had a tolerance threshold for PfAlba1-Ty1 levels. When we treated 3D7+PfAlba1-Ty1 parasites that were initially growing with 2.5 μg/ml blasticidin-S (BS) in the growth medium with increasing amounts of BS for two days, PfAlba1-Ty1 protein levels gradually increased (Fig. 2a), reaching a maximum of fivefold upregulation at 20 μg/ml BS relative to 2.5 μg/ml BS (Fig. 2b). We also observed that the levels of endogenous PfAlba1 increased in a BS dose-dependent manner (Fig. 2a; Figure S2a in Additional file 1), but not of a second Alba family protein PfAlba4 (Fig. 2a) and two other orthologs, PfAlba2 and PfAlba3 (data not shown), indicating that overexpression of PfAlba1-Ty1 directly affected endogenous PfAlba1 levels. Note that, even at 2.5 μg/ml BS, PfAlba1-Ty1 transfectants express higher levels of total PfAlba1 compared with the empty vector control (Figs. 1b and 2a).Fig. 2

Bottom Line: Using deep sequencing of affinity-purified PfAlba1-associated RNAs, we identify 1193 transcripts that directly bind to PfAlba1 in trophozoites.For 105 such transcripts, 43 % of which are uncharacterized and 13 % of which encode erythrocyte invasion components, the steady state levels significantly change at this stage, evidencing a role for PfAlba1 in maintaining mRNA homeostasis.This mode of post-transcriptional regulation may be especially important for P. falciparum erythrocyte invasion components that have to be assembled into apical secretory organelles in a highly time-dependent manner towards the end of the parasite's asexual lifecycle.

View Article: PubMed Central - PubMed

Affiliation: Unité Biologie des Interactions Hôte-Parasite, Département de Parasites et Insectes Vecteurs, Institut Pasteur, Paris, 75015, France. shruthi-sridhar.vembar@pasteur.fr.

ABSTRACT

Background: Transcriptome-wide ribosome occupancy studies have suggested that during the intra-erythrocytic lifecycle of Plasmodium falciparum, select mRNAs are post-transcriptionally regulated. A subset of these encodes parasite virulence factors required for invading host erythrocytes, and are currently being developed as vaccine candidates. However, the molecular mechanisms that govern post-transcriptional regulation are currently unknown.

Results: We explore the previously identified DNA/RNA-binding protein PfAlba1, which localizes to multiple foci in the cytoplasm of P. falciparum trophozoites. We establish that PfAlba1 is essential for asexual proliferation, and subsequently investigate parasites overexpressing epitope-tagged PfAlba1 to identify its RNA targets and effects on mRNA homeostasis and translational regulation. Using deep sequencing of affinity-purified PfAlba1-associated RNAs, we identify 1193 transcripts that directly bind to PfAlba1 in trophozoites. For 105 such transcripts, 43 % of which are uncharacterized and 13 % of which encode erythrocyte invasion components, the steady state levels significantly change at this stage, evidencing a role for PfAlba1 in maintaining mRNA homeostasis. Additionally, we discover that binding of PfAlba1 to four erythrocyte invasion mRNAs, Rap1, RhopH3, CDPK1, and AMA1, is linked to translation repression in trophozoites whereas release of these mRNAs from a PfAlba1 complex in mature stages correlates with protein synthesis.

Conclusions: We show that PfAlba1 binds to a sub-population of asexual stage mRNAs and fine-tunes the timing of translation. This mode of post-transcriptional regulation may be especially important for P. falciparum erythrocyte invasion components that have to be assembled into apical secretory organelles in a highly time-dependent manner towards the end of the parasite's asexual lifecycle.

No MeSH data available.


Related in: MedlinePlus