Final Report Summary - PFALBA (Role of the PfAlbas, a novel DNA and RNA binding protein family, in Plasmodium falciparum chromatin biology and RNA regulation)
Plasmodium falciparum, the causal agent of the most deadly form of malaria, undergoes complex life cycle transitions in the mosquito vector and human host. Pathology of P. falciparum infection manifests during the 48-hour asexual, intra-erythrocytic developmental cycle (IDC), which is comprised of three morphologically and metabolically distinct stages: ring (0-24 h), trophozoite (24-38 h) and schizont (38-48 h). Additionally, a small percentage of parasites generated during the IDC commit to sexual development and subsequent transmission to the mosquito vector (ref). Notably, vaccine production against the asexual stages has not met with great success (ref), and the parasite is evolving resistance to currently available anti-malarial drugs (ref). Therefore, a comprehensive understanding of essential parasite processes such as gene regulation is imperative to eradicate malaria.
Of interest to this study is the DNA/RNA-binding PfAlba family. Alba domain-containing proteins have been characterized to act on DNA and RNA in archaea and other parasites. For example, Alba binding in the parasite Toxoplasma gondii modulates the translation of select mRNAs, including the Alba mRNAs, and P. berghei Albas associate with a translation repression complex in sexual stages, though their exact function is unknown. In P. falciparum, we previously established that PfAlba1-4 bind to RNA in vitro and that PfAlba1, 2 and 4 localize to punctate loci in the cytoplasm of trophozoites and schizonts, reminiscent of RNA storage/processing centers. We therefore hypothesized that some of the PfAlbas would play key roles in cytoplasmic post-transcriptional gene regulation during the P. falciparum IDC. To explore this, we focused on PfAlba1, a 27 kDa protein which has two RNA-binding domains: an N-terminal Alba domain that is conserved amongst all PfAlbas and a C-terminal arginine/glycine-rich (RGG) domain. We found that PfAlba1 overexpression resulted in an inhibition of parasite intra-erythrocytic growth and perturbed the steady state transcriptome of trophozoite stages, a stage at which the protein is predominantly cytoplasmic. To understand this phenotype, we identified PfAlba1’s mRNA targets using RNA immunoprecipitation and in vitro pulldowns followed by high throughput sequencing. For select transcripts, we found that PfAlba1 binding resulted in translation repression, with release from the PfAlba1 complex coincident with efficient translation. In conclusion, our results point to a role of PfAlba1 in maintaining the correct timing of translation of specific transcripts. This is the first study to show that an RNA-binding protein sequesters select non-cognate mRNAs and regulates their translation in P. falciparum asexual stages.
We have also begun characterizing PfAlba2-4 and find that these proteins have non-overlapping functions during the IDC. For example, we were unable to knockout or knockdown these proteins and found that exogenous overexpression of PfAlba2/3-Ty1, but not PfAlba4-Ty1, resulted in a strong growth retardation phenotype. Using high throughput sequencing, we analyzed the steady state transcriptomes of these parasites and found that the trophozoite transcriptome of PfAlba2/3-Ty1 parasites appeared to be more schizont-like. This suggested that PfAlba2/3 may be involved in regulating the cell cycle of the parasite, at least at the RNA level, without affecting parasite morphology; this was assessed by flow cytometry and GIEMSA staining of the parasite cultures. Of note, PfAlba4 overexpession does not cause growth or transcriptomic phenotypes. We therefore plan to investigate the nuclear/chromatin-regulatory role of this protein by using a knockdown strategy, and this is in progress.
Overall, our analyses point to an essential role for the PfAlbas in P. falciparum blood stage development, in particular in the regulation of RNA biology. We therefore want to comprehensively characterize the RNA-binding preferences of the PfAlbas in vitro. Using recombinant proteins – for full-length PfAlbas as well as individual domains of each PfAlba – we are performing Systematic Evolution of Ligands by EXponential enrichment (i.e. SELEX) with random RNA oligonucleotide libraries to identify aptamer sequences that are specifically bound by the PfAlbas. We postulate that the RNA aptamers identified in this study can be developed as anti-malarials. Such a therapeutic strategy is already being utilized to treat several disorders including angiomas, acute myeloid leukemia, renal cell carcinoma, macular degeneration, choroidal neovascularization, etc. We also plan to expand our biochemical studies to P. vivax Albas, currently a black box, and whose characterization will be important to determine the specificity of the anti-Alba RNA aptamers.
Of interest to this study is the DNA/RNA-binding PfAlba family. Alba domain-containing proteins have been characterized to act on DNA and RNA in archaea and other parasites. For example, Alba binding in the parasite Toxoplasma gondii modulates the translation of select mRNAs, including the Alba mRNAs, and P. berghei Albas associate with a translation repression complex in sexual stages, though their exact function is unknown. In P. falciparum, we previously established that PfAlba1-4 bind to RNA in vitro and that PfAlba1, 2 and 4 localize to punctate loci in the cytoplasm of trophozoites and schizonts, reminiscent of RNA storage/processing centers. We therefore hypothesized that some of the PfAlbas would play key roles in cytoplasmic post-transcriptional gene regulation during the P. falciparum IDC. To explore this, we focused on PfAlba1, a 27 kDa protein which has two RNA-binding domains: an N-terminal Alba domain that is conserved amongst all PfAlbas and a C-terminal arginine/glycine-rich (RGG) domain. We found that PfAlba1 overexpression resulted in an inhibition of parasite intra-erythrocytic growth and perturbed the steady state transcriptome of trophozoite stages, a stage at which the protein is predominantly cytoplasmic. To understand this phenotype, we identified PfAlba1’s mRNA targets using RNA immunoprecipitation and in vitro pulldowns followed by high throughput sequencing. For select transcripts, we found that PfAlba1 binding resulted in translation repression, with release from the PfAlba1 complex coincident with efficient translation. In conclusion, our results point to a role of PfAlba1 in maintaining the correct timing of translation of specific transcripts. This is the first study to show that an RNA-binding protein sequesters select non-cognate mRNAs and regulates their translation in P. falciparum asexual stages.
We have also begun characterizing PfAlba2-4 and find that these proteins have non-overlapping functions during the IDC. For example, we were unable to knockout or knockdown these proteins and found that exogenous overexpression of PfAlba2/3-Ty1, but not PfAlba4-Ty1, resulted in a strong growth retardation phenotype. Using high throughput sequencing, we analyzed the steady state transcriptomes of these parasites and found that the trophozoite transcriptome of PfAlba2/3-Ty1 parasites appeared to be more schizont-like. This suggested that PfAlba2/3 may be involved in regulating the cell cycle of the parasite, at least at the RNA level, without affecting parasite morphology; this was assessed by flow cytometry and GIEMSA staining of the parasite cultures. Of note, PfAlba4 overexpession does not cause growth or transcriptomic phenotypes. We therefore plan to investigate the nuclear/chromatin-regulatory role of this protein by using a knockdown strategy, and this is in progress.
Overall, our analyses point to an essential role for the PfAlbas in P. falciparum blood stage development, in particular in the regulation of RNA biology. We therefore want to comprehensively characterize the RNA-binding preferences of the PfAlbas in vitro. Using recombinant proteins – for full-length PfAlbas as well as individual domains of each PfAlba – we are performing Systematic Evolution of Ligands by EXponential enrichment (i.e. SELEX) with random RNA oligonucleotide libraries to identify aptamer sequences that are specifically bound by the PfAlbas. We postulate that the RNA aptamers identified in this study can be developed as anti-malarials. Such a therapeutic strategy is already being utilized to treat several disorders including angiomas, acute myeloid leukemia, renal cell carcinoma, macular degeneration, choroidal neovascularization, etc. We also plan to expand our biochemical studies to P. vivax Albas, currently a black box, and whose characterization will be important to determine the specificity of the anti-Alba RNA aptamers.