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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

Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite (T) or schizont (S) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. cLeft panel: IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e., T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites (T) and schizonts (S) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies
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Fig7: Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite (T) or schizont (S) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. cLeft panel: IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e., T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites (T) and schizonts (S) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies

Mentions: Finally, to investigate the molecular basis of the fine-tuning of translation of erythrocyte invasion transcripts, we assessed the association of RhopH3, CDPK1, Clag9, Rap1, Rap2, AMA1, and TRAMP mRNAs with PfAlba1-Ty1 during typical protein expression, i.e., the schizont stage (42–45 h), and approximately 14–17 h before detectable protein expression, i.e., trophozoites (28–30 h) (Fig. 7a). We hypothesized that invasion transcripts that are bound to and translationally repressed by PfAlba1 in trophozoites would be released from PfAlba1 in schizonts to allow for translation. We first IPed PfAlba1-Ty1 with anti-Ty1 antibodies under non-denaturing conditions (Fig. 7b), and performed qRT-PCR analysis with primers specific to the selected invasion transcripts. We included FIKK7.1 and FIKK9.6 as upregulated, but unbound, controls and analyzed three unchanged and unbound mRNAs as controls for immunoprecipitation: aldolase, actin I and seryl tRNA synthetase. For each gene, we also determined the fold change in transcript levels from trophozoite to schizont stages relative to actin I mRNA (Figure S8 in Additional file 1) and used this value to calculate the fold change in association from trophozoites to schizonts.Fig. 7


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)

Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite (T) or schizont (S) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. cLeft panel: IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e., T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites (T) and schizonts (S) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig7: Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite (T) or schizont (S) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. cLeft panel: IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e., T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites (T) and schizonts (S) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies
Mentions: Finally, to investigate the molecular basis of the fine-tuning of translation of erythrocyte invasion transcripts, we assessed the association of RhopH3, CDPK1, Clag9, Rap1, Rap2, AMA1, and TRAMP mRNAs with PfAlba1-Ty1 during typical protein expression, i.e., the schizont stage (42–45 h), and approximately 14–17 h before detectable protein expression, i.e., trophozoites (28–30 h) (Fig. 7a). We hypothesized that invasion transcripts that are bound to and translationally repressed by PfAlba1 in trophozoites would be released from PfAlba1 in schizonts to allow for translation. We first IPed PfAlba1-Ty1 with anti-Ty1 antibodies under non-denaturing conditions (Fig. 7b), and performed qRT-PCR analysis with primers specific to the selected invasion transcripts. We included FIKK7.1 and FIKK9.6 as upregulated, but unbound, controls and analyzed three unchanged and unbound mRNAs as controls for immunoprecipitation: aldolase, actin I and seryl tRNA synthetase. For each gene, we also determined the fold change in transcript levels from trophozoite to schizont stages relative to actin I mRNA (Figure S8 in Additional file 1) and used this value to calculate the fold change in association from trophozoites to schizonts.Fig. 7

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