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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

Eleven percent of the transcripts deregulated in PfAlba1-Ty1 trophozoites directly bind to PfAlba1, with binding resulting in the translational repression of select components of the erythrocyte invasion machinery. a Venn diagrams were used to represent the overlap of transcripts identified in the co-IPed (co-IP; Additional file 2), in vitro-bound (In Vitro; Additional file 4), and differentially regulated (Exp.; Additional file 6) datasets. b Pie chart showing the functionally over-represented categories in the 105 PfAlba1-bound and deregulated transcripts. c Bootstrap hierarchical clustering was used to partition 14 transcripts upregulated in PfAlba1-Ty1 trophozoites based on their binding (log2(fold change); blue shading) to PfAlba1-Ty1 (co-IP) and GST-PfAlba1 (In Vitro). The numbers in the boxes are the bootstrap scores. The resulting clusters were PfAlba1-bound (upper cluster) and PfAlba1-unbound (lower cluster). dTop panel: qRT-PCR analysis of the indicated genes was performed using mRNA isolated from empty vector or PfAlba1-Ty1 trophozoites. The y-axis denotes -ΔΔCt, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). Bottom panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies. Lysates prepared from either 3D7 rings (R ctrl) or schizonts (S ctrl) served as a control for protein detection, except for PfFIKK7.1. ** = Exceptions to the observed correlation between PfAlba1-RNA binding and translational repression
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Fig6: Eleven percent of the transcripts deregulated in PfAlba1-Ty1 trophozoites directly bind to PfAlba1, with binding resulting in the translational repression of select components of the erythrocyte invasion machinery. a Venn diagrams were used to represent the overlap of transcripts identified in the co-IPed (co-IP; Additional file 2), in vitro-bound (In Vitro; Additional file 4), and differentially regulated (Exp.; Additional file 6) datasets. b Pie chart showing the functionally over-represented categories in the 105 PfAlba1-bound and deregulated transcripts. c Bootstrap hierarchical clustering was used to partition 14 transcripts upregulated in PfAlba1-Ty1 trophozoites based on their binding (log2(fold change); blue shading) to PfAlba1-Ty1 (co-IP) and GST-PfAlba1 (In Vitro). The numbers in the boxes are the bootstrap scores. The resulting clusters were PfAlba1-bound (upper cluster) and PfAlba1-unbound (lower cluster). dTop panel: qRT-PCR analysis of the indicated genes was performed using mRNA isolated from empty vector or PfAlba1-Ty1 trophozoites. The y-axis denotes -ΔΔCt, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). Bottom panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies. Lysates prepared from either 3D7 rings (R ctrl) or schizonts (S ctrl) served as a control for protein detection, except for PfFIKK7.1. ** = Exceptions to the observed correlation between PfAlba1-RNA binding and translational repression

Mentions: Having identified PfAlba1’s in vivo and in vitro RNA interactome and RNAs that are differentially regulated when parasites produce excess PfAlba1, we posited that RNAs detected in all of these datasets would likely be the first set of transcripts targeted by PfAlba1 for post-transcriptional regulation. Accordingly, we outlined their intersection using a Venn diagram and found that a small proportion, 8.8 % (n = 105), of the 1193 transcripts that bound in vivo and in vitro was twofold or more differentially regulated: 97 upregulated and 8 downregulated (Fig. 6a). Functional group analysis of the 105 transcripts revealed that 43 % encoded conserved proteins of unknown function, while the remaining encoded 13 components of the invasion machinery, such as PfCDPK1, PfEBA175, PfRhopH2, PfRhopH3, PfRON2, PfRON3, PfRON4, and PfRh2a and 2b, 14 kinases and related signaling molecules, three actin- and myosin-related proteins, and three ApiAP2 proteins, to name a few (Fig. 6b; Additional file 8). Additionally, the differentially regulated transcriptome contained 38 transcripts that only bound in vivo and 119 transcripts that only bound in vitro (Fig. 6a; Additional file 8), expanding the primary list of mRNAs targeted by PfAlba1 complexes for regulation during the IDC to 262. To visualize the cellular and functional distribution of annotated genes in the Venn intersections of Fig. 6a, we designed a schematic of a P. falciparum asexual blood stage cell and mapped each gene and its intersection category onto the schematic using MapMan [30] (Figure S7 in Additional file 1). It became apparent that mRNAs encoding proteins that contribute to erythrocyte invasion, including components of apical organelles and the actin- and myosin-related motor proteins, constitute a significant portion of PfAlba1 targets.Fig. 6


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)

Eleven percent of the transcripts deregulated in PfAlba1-Ty1 trophozoites directly bind to PfAlba1, with binding resulting in the translational repression of select components of the erythrocyte invasion machinery. a Venn diagrams were used to represent the overlap of transcripts identified in the co-IPed (co-IP; Additional file 2), in vitro-bound (In Vitro; Additional file 4), and differentially regulated (Exp.; Additional file 6) datasets. b Pie chart showing the functionally over-represented categories in the 105 PfAlba1-bound and deregulated transcripts. c Bootstrap hierarchical clustering was used to partition 14 transcripts upregulated in PfAlba1-Ty1 trophozoites based on their binding (log2(fold change); blue shading) to PfAlba1-Ty1 (co-IP) and GST-PfAlba1 (In Vitro). The numbers in the boxes are the bootstrap scores. The resulting clusters were PfAlba1-bound (upper cluster) and PfAlba1-unbound (lower cluster). dTop panel: qRT-PCR analysis of the indicated genes was performed using mRNA isolated from empty vector or PfAlba1-Ty1 trophozoites. The y-axis denotes -ΔΔCt, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). Bottom panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies. Lysates prepared from either 3D7 rings (R ctrl) or schizonts (S ctrl) served as a control for protein detection, except for PfFIKK7.1. ** = Exceptions to the observed correlation between PfAlba1-RNA binding and translational repression
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587749&req=5

Fig6: Eleven percent of the transcripts deregulated in PfAlba1-Ty1 trophozoites directly bind to PfAlba1, with binding resulting in the translational repression of select components of the erythrocyte invasion machinery. a Venn diagrams were used to represent the overlap of transcripts identified in the co-IPed (co-IP; Additional file 2), in vitro-bound (In Vitro; Additional file 4), and differentially regulated (Exp.; Additional file 6) datasets. b Pie chart showing the functionally over-represented categories in the 105 PfAlba1-bound and deregulated transcripts. c Bootstrap hierarchical clustering was used to partition 14 transcripts upregulated in PfAlba1-Ty1 trophozoites based on their binding (log2(fold change); blue shading) to PfAlba1-Ty1 (co-IP) and GST-PfAlba1 (In Vitro). The numbers in the boxes are the bootstrap scores. The resulting clusters were PfAlba1-bound (upper cluster) and PfAlba1-unbound (lower cluster). dTop panel: qRT-PCR analysis of the indicated genes was performed using mRNA isolated from empty vector or PfAlba1-Ty1 trophozoites. The y-axis denotes -ΔΔCt, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). Bottom panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies. Lysates prepared from either 3D7 rings (R ctrl) or schizonts (S ctrl) served as a control for protein detection, except for PfFIKK7.1. ** = Exceptions to the observed correlation between PfAlba1-RNA binding and translational repression
Mentions: Having identified PfAlba1’s in vivo and in vitro RNA interactome and RNAs that are differentially regulated when parasites produce excess PfAlba1, we posited that RNAs detected in all of these datasets would likely be the first set of transcripts targeted by PfAlba1 for post-transcriptional regulation. Accordingly, we outlined their intersection using a Venn diagram and found that a small proportion, 8.8 % (n = 105), of the 1193 transcripts that bound in vivo and in vitro was twofold or more differentially regulated: 97 upregulated and 8 downregulated (Fig. 6a). Functional group analysis of the 105 transcripts revealed that 43 % encoded conserved proteins of unknown function, while the remaining encoded 13 components of the invasion machinery, such as PfCDPK1, PfEBA175, PfRhopH2, PfRhopH3, PfRON2, PfRON3, PfRON4, and PfRh2a and 2b, 14 kinases and related signaling molecules, three actin- and myosin-related proteins, and three ApiAP2 proteins, to name a few (Fig. 6b; Additional file 8). Additionally, the differentially regulated transcriptome contained 38 transcripts that only bound in vivo and 119 transcripts that only bound in vitro (Fig. 6a; Additional file 8), expanding the primary list of mRNAs targeted by PfAlba1 complexes for regulation during the IDC to 262. To visualize the cellular and functional distribution of annotated genes in the Venn intersections of Fig. 6a, we designed a schematic of a P. falciparum asexual blood stage cell and mapped each gene and its intersection category onto the schematic using MapMan [30] (Figure S7 in Additional file 1). It became apparent that mRNAs encoding proteins that contribute to erythrocyte invasion, including components of apical organelles and the actin- and myosin-related motor proteins, constitute a significant portion of PfAlba1 targets.Fig. 6

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