Browsing by Author "Chi, Jen-Tsan Ashley"

The physiology of the tumor microenvironment is characterized by lower oxygen (hypoxia), higher lactate, extracellular acidosis and glucose starvation. We examined the global, transcriptional cellular responses to each of these microenvironmental stresses in vitro, projected them onto clinical breast cancer patients' samples in vivo, and returned to perform further in vitro experiments to investigate the potential mechanisms involved in these stress responses. The reciprocal exchange of information was critical and advanced our understanding of the potential clinical relevance of cellular responses.

Our expression array result showed that lactic acidosis induces a strong response, distinct from that of hypoxia in human mammalian epithelial cells (HMECs), indicating lactic acidosis is not only a by-product of hypoxia but has a unique role as a stimulant to cells in the tumor microenvironment. Cellular responses to lactosis and acidosis further demonstrated that acidosis was the main driving force in the lactic acidosis response. These responding gene signatures were then statistically projected into clinical breast cancer patients' expression data sets. The hypoxia response, as reported previously, was associated with bad prognosis, where as the lactic acidosis and acidosis responses, were associated with good prognosis. Additionally, the acidosis response could be used to separate breast tumors with high versus low aggressiveness based on its inversed correlation with metastatic character. We further discovered that lactic acidosis, in contrast to hypoxia, abolished Akt signaling. Moreover, it downregulated glycolysis and shifted energy utilization towards aerobic respiration.

We continued to examine the cellular response to lactic acidosis temporally in MCF7 cells, a breast cancer cell line. The lactic acidosis response of MCF7 cells also showed the prognostic result of better clinical outcomes in datasets of breast cancer patients. The lactic acidosis responses of HMEC and MCF cells were highly correlated. Strikingly in MCF7 cells, lactic acidosis and glucose deprivation actually induced similar transcriptional profiles, with only a few genes being oppositely regulated. Furthermore, lactic acidosis, similar to glucose starvation, induced AMPK signaling and abolished mTOR. However, lactic acidosis and glucose deprivation induced opposite glucose uptake phenotypes. Lactic acidosis significantly repressed glucose uptake whereas glucose deprivation significantly induced it. Among the genes differentially regulated by these two stresses, thioredoxin-interacting protein (TXNIP) was among the most different. The negative regulatory role of TXNIP on glucose uptake has been demonstrated previously. In the cancer research field, TXNIP is recognized as a tumor suppressor gene. We observed that lactic acidosis induced TXNIP strongly and most importantly, TXNIP played a critical role in regulating glucose uptake in cells under lactic acidosis. Furthermore, MondoA, the transcription factor and glucose sensor previously reported to regulate TXNIP induction upon glucose exposure, was also responsible for regulating TXNIP under lactic acidosis. We demonstrated that TXNIP not only plays an important role in the lactic acidosis response but also has strong prognostic power to separate breast cancer patients based on survival.

Dengue viruses (DENV) and other mosquito-borne flaviviruses are rapidly emerging human pathogens that threaten nearly half of the world’s population. There is currently no effective vaccine or antiviral therapeutics for the prophylaxis or treatment of DENV. While traditional drug development efforts have focused on inhibitors of viral enzymes, an alternative approach is to target host proteins that support virus replication. In an effort to identify novel human enzymes important for the DENV-2 life-cycle, we conducted a genome-wide RNAi screen and identified ERI3, a putative 3′-5′ exonuclease, as a novel DENV-2 host factor. Cell-free assays confirmed that purified ERI3 is capable of degrading single-stranded RNA in a 3′ to 5′ direction. We conducted a screen for compounds that inhibit ERI3 in vitro and identified small molecules that antagonized both exonuclease activity. In summary, we identified a host exonuclease that is important for DENV-2 replication and is a potential therapeutic target. Our approach illustrates the utility of identifying host enzymatic functions for development of anti-viral drugs.

Large-scale molecular epidemiologic studies of Plasmodium falciparum parasites have provided insights into parasite biology and transmission, can identify the spread of drug resistance, and are useful in assessing vaccine targets. The polyclonal nature infections in high transmission settings is problematic for traditional genotyping approaches. Next-generation sequencing approaches to parasite genotyping allow sensitive detection of minority variants, disaggregation of complex parasite mixtures, and scalable processing of large samples sets. Therefore, we designed, validated, and applied to field parasites a new approach that leverages sequencing of individually barcoded samples in a multiplex manner. We utilize variant barcodes, invariant linker sequences and modular template-specific primers to allow for the simultaneous generation of high-dimensional sequencing data of multiple gene targets. This modularity permits a cost-effective and reproducible way to query many genes at once. In mixtures of reference parasite genomes, we quantitatively detected unique haplotypes comprising as little as 2% of a polyclonal infection. We applied this genotyping approach to field-collected parasites collected in Western Kenya in order to simultaneously obtain parasites genotypes at three unlinked loci. In summary, we present a rapid, scalable, and flexible method for genotyping individual parasites that enables molecular epidemiologic studies of parasite evolution, population structure and transmission.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. Additionally, cancer cells exposed to these stresses are more resistant to therapies, more likely to metastasize and often are worse for patient prognosis. While the presence of these stresses is generally negative for cancer patients, since these stresses are mostly unique to the TME, they also offer an opportunity to develop more selective therapeutics. If we achieve a better understanding of the adaptive mechanisms cancer cells employ to survive the TME stresses, then hopefully we, as a scientific community, can devise more effective cancer therapeutics specifically targeting cancer cells under stress. To systematically identify genes that modulate cancer cell survival under stresses, we performed shRNA screens under hypoxia or lactic acidosis. From these screens, we discovered that genetic depletion of acetyl-CoA carboxylase alpha (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Furthermore, the loss of ACLY or ACC1 reduced the levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate was paradoxically increased under hypoxia when ACC1 or ACLY were depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulated the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

The development of cancer is a process by which an accumulation of genetic changes leads to uncontrolled replication of cells. Since the process of mutation is random, the set of alterations that occur and accumulate during tumorigenesis in one individual is different from that of another. These genetic differences drive tumor heterogeneity. One of the first technologies used to explore genome-wide heterogeneity was the microarray, which can be used to measure the expression of tens of thousands of genes. By exploring differences in expression of not just single genes, but groups of genes that may be altered in one set of tumors compared to another, researchers were able to classify subtypes of cancer that had relevance in disease aggressiveness, treatment, and prognosis. Furthermore, by looking at genome-wide patterns of expression, it is possible to identify specific oncogenic pathways that are activated and critical in driving tumor cell survival, growth, or metastasis. My research utilizes the patterns of expression derived from microarray analyses to study tumor heterogeneity, particularly in response to targeted cancer therapy, and mechanisms of cell death following oncogenic deregulation.

One of the cancer types that has been explored through expression array analysis is B-cell lymphoma. Human aggressive B-cell non-Hodgkin lymphomas (NHL) encompass the continuum between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), and display considerable clinical and biologic heterogeneity, most notably related to therapy response. We previously showed that lymphomas arising in the E&mu-Myc transgenic mouse are heterogeneous, mirroring genomic differences between BL and DLBCL. Given the clinical heterogeneity in NHL and the need to develop strategies to match therapeutics with discrete forms of disease, we investigated the extent to which genomic variation in the E&mu-Myc model predicts response to therapy. We used genomic analyses to classify E&mu-Myc lymphomas, link E&mu-Myc lymphomas with NHL subtypes, and identify lymphomas with predicted resistance to conventional and NF-&kappaB targeted therapies. Experimental evaluation of these predictions links genomic profiles with distinct outcomes to conventional and targeted therapies in the E&mu-Myc model, and establishes a framework to test novel targeted therapies or combination therapies in specific genomically-defined lymphoma subgroups. In turn, this will rationally inform the design of new treatment options for aggressive human NHL.

The second aspect of my thesis looks at the mechanisms of apoptosis following oncogene deregulation. The Rb-E2F pathway is a critical oncogenic pathway that is frequently mutated in cancers. Alterations in the pathway affect genome-wide expression in the cell, which in turn lead to deregulation of the cell cycle. The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors that cooperate with E2F to mediate the specificity of E2F function. In this work, we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

Taken together, these studies highlight the importance and power of analyzing genome-wide patterns of expression in investigating cancer heterogeneity, its ability to help predict treatment response, and its role in discovering the mechanisms behind the consequences of gene deregulation.

All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of “alarmone” (p)ppGpp to arrest proliferation and reprogram the transcriptome. While mammalian genomes encode MESH1—the homologue of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and is associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumorsphere formation, and retarded xenograft growth. These anti-tumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed mRNA expression of TAZ, a HIPPO co-activator mediating the expression of many proliferative genes. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, sphere formation inhibition, dNTP depletion, retarded xenograft growth and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.

O-GlcNAcylation is a reversible post-translational modification that decorates an O-linked ß-N-acetylglucosamine (O-GlcNAc) moiety onto the serine/threonine residues of target proteins. In mammals, this modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser). Several studies have revealed that O-GlcNAcylation can be responsive to metabolic status or stress stimulation. However, the specific O-GlcNAc targets in response to various nutrient and stress signals are not well defined. We conducted a global transcriptome profiling in triple-negative breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. Unexpectedly, we found that the NRF2-dependent stress response positively correlates with lower OGT activity in multiple human tumor gene expression datasets. NRF2, a major transcriptional regulator of redox balance, is usually activated by oxidative stress but degraded by proteasome under basal conditions via the KEAP1-CUL3 ubiquitin ligase-mediated polyubiquitination. Using azidosugar metabolic labeling, bioorthogonal chemistry and mass spectrometry, we determined that the NRF2 negative regulator KEAP1 is O-GlcNAcylated within its BTB and Kelch motifs. KEAP1 belongs to the Kelch-like (KLHL) adaptor protein family, which was known to regulate substrate proteostasis via CUL3-mediated ubiquitination. Of 11 candidate O-GlcNAc sites on KEAP1, serine 104 is responsible for regulating NRF2 activity by promoting the KEAP1-CUL3 interaction. Interestingly, we found that other KLHL protein, gigaxonin, is also O-GlcNAcylated on up to nine candidate sites. Mutation of gigaxonin is known to cause giant axonal neuropathy (GAN), a neurodegenerative disease that is characterized by the accumulation of intermediate filaments in axons. We found gigaxonin O-GlcNAcylation is required for its ability to facilitate the ubiquitination and proteolysis of intermediate filaments. Mutation of specific gigaxonin O-GlcNAcylation sites compromised its optimal interactions with intermediate filament proteins. This finding provides new molecular insight into GAN pathogenesis. The link between proteostasis and nutrient-sensing is fundamentally important yet incompletely understood. Together, my dissertation work has revealed new connections among nutrient-sensitive glycosylation, KLHL protein function, proteostasis and downstream signaling, with relevance for human diseases.

Here, I sought to determine the molecular mechanisms of the cellular response to stresses in two contexts. In the first part of my thesis, I focus on how ferroptosis, a lipid oxidative stress-induced cell death, can be regulated by cell density via an evolutionarily conserved pathway effector. In the second part, I focus on the transcriptional response of red blood cells (RBCs) during the refrigerated storage.

Ferroptosis is a novel form of programmed cell death characterized by the accumulation of lipid peroxidation. It can be induced by the oxidative stress caused by starvation of cystine, inhibition of glutathione peroxidase 4, or activation of NADPH oxidase(s). The canonical ferroptosis inducer, erastin, is a small molecule which triggers oxidative stress by inhibiting the cystine-glutamate transporter (xCT) and thus reduces intracellular cysteine level and glutathione biosynthesis. Recent studies indicate ferroptosis may have therapeutic potential toward cancer. However, much remains unknown about the determinants of ferroptosis susceptibility. We observed that vulnerability to the ferroptosis of cancer cells is highly influenced by cell confluency. Since cell density can be sensed by the evolutionarily conserved Hippo pathway effectors, YAP/TAZ, we hypothesize if these Hippo pathway effectors are involved in erastin-induced ferroptosis response. My data show that TAZ, instead of YAP, is abundantly expressed in both renal and ovarian cancer cells and undergoes density-dependent nuclear/cytosolic translocation. TAZ removal confers ferroptosis resistance, while overexpression of constitutively active form of TAZ, TAZS89A, sensitizes cells to ferroptosis. Similarly, I found that a lower TAZ level in the recurrent ovarian cancer is responsible for reduced ferroptosis susceptibility of these cells. I further investigated the mechanisms by which TAZ regulates ferroptosis. and found that TAZ regulates ferroptosis through EMP1-NOX4 axis in renal cancers and ANGPTL4-NOX2 axis in ovarian cancers. The relevance of the Hippo pathway effector with ferroptosis suggests that ferroptosis-inducing agents may be used to target the TAZ-activated tumors.

The second part of my dissertation investigated the molecular mechanisms of transcriptome changes inside RBCs during ex vivo storages. RBCs are the major component of blood transfusions, one of the most common procedures in the hospital. In addition, some athletes utilize blood transfusion of stored RBCs to increase athletic performance, a practice banned by the world anti-doping agency. Currently, RBCs can be stored for up to 42 days at ~4°C before transfusion. However, transfusion with RBCs after long storage duration may correlate with a poorer prognosis compared with fresh RBCs and results in increased morbidity and mortality. To recognize the undesirable effects of prolonged RBC storage on transfusion recipients, it is critical to understand storage-associated RBC changes. To this end, our lab has previously identified a variety of RNA species in mature RBCs and profiled the miRNA changes that occur in RBCs at different time intervals during in vitro storage. This profiling demonstrates that the abundance of most RBC miRNAs did not change significantly during the 42 days of refrigerated storage, indicating extremely long decay half-lives. Unexpectedly, miR-720, a cleavage product of tRNAThr, increased dramatically in the first two weeks and persisted during storage. Furthermore, I present evidence for a role of angiogenin in tRNA cleavage to generate miR-720 during RBC storage. The dramatic increase in miR-720 may be used to monitor transfused RBCs in clinical patients, athletes performing blood doping, and other settings. Additionally, the increase in miR-720 levels in the stored RBC may potentially contribute to the cellular and clinical phenotypes associated with storage lesions.

Taken together, these studies on how human cells respond to stresses have the potentials as guidance for cancer patients toward ferroptosis-inducing chemotherapeutics or provide a novel way of detecting blood doping or understanding the RBC storage lesions.

In this dissertation, I will present two studies on the non-canonical role of the how two kinases participate in the oncogenesis via non-canonical mechanisms. In the first study, we investigate the role of CoA synthase (COASY) in the regulation of the protein acetylation during mitosis. In the second study, we will investigate the dysregulation of the RIPK3 in the recurrent tumor cells render them uniquely susceptible to death signaling triggered by cystine deprivation.

The temporal activation of kinases and timely ubiquitin-mediated degradation is central to faithful mitosis. However, whether acetylation of mitotic proteins are involved in regulation mechanism of mitosis is relatively rarely studied. Here we present evidence that acetylation controlled by COASY and acetyltransferase CBP constitutes a novel mechanism that ensures faithful mitosis. We found that COASY knockdown triggers prolonged mitosis and multinucleation. Acetylome analysis reveals that COASY inactivation leads to hyper-acetylation of a subset of proteins associated with mitosis, including CBP and an Aurora A kinase activator, TPX2. We found that TPX2 is hyper-acetylated under COASY knockdown, which rendered TPX2 accumulation by resistant to ubiquitination. Further experiments confirmed that the direct interaction of COASY and CBP regulates CBP-mediated TPX2 acetylation. We therefore propose a regulatory mechanism that a transient CBP-mediated TPX2 acetylation is associated with TPX2 accumulation and Aurora A activation during early mitosis. The recruitment of COASY inhibits CBP-mediated TPX2 acetylation, promoting TPX2 degradation for mitotic exit. Remarkably, pharmacological and genetic inactivation of CBP effectively rescued the mitotic defects caused by COASY knockdown. We also found that PPAT domain on COASY is in responsible for the inhibitory effect of COASY on CBP. Together, our findings uncover a novel mitotic regulation wherein COASY and CBP coordinate an acetylation network to enforce productive mitosis.

Tumor recurrence results in most of the mortality in breast cancer. Recurrent tumor cells are usually incurable and unresponsive to most of the treatments. We have previous reported that triple negative breast cancer cells are more sensitive to Cystine deprivation though epithelial-mesenchymal transition. Here we report that recurrent tumor cells are more sensitive to Cystine deprivation when comparing to primary tumor cells. RNA expression profiling identifies a much higher expression of RIPK3 RNA in recurrent cells. RIPK3 silencing in recurrent cells can rescue the programmed necrosis under Cystine deprivation. We further found that RIPK3 is crucial for the rapid cell proliferation in recurrent cells. Thus, the collateral vulnerability can be a potential therapeutic target for recurrent tumor specific treatment. We are still actively investigating the molecular mechanism for the recurrent cells taking advantage of high RIPK3 expression for rapid cell growth.

Ferroptosis is a form of regulated cell death featured by lipid peroxidation and breakage of cell membrane. However, the molecular mediators and regulators are not fully understood. Here, we identified the metazoan homologues of ppGpp hydrolase (MESH1) is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity that is captured by the crystal structure of the MESH1-NADPH complex. Ferroptosis elevates MESH1, whose upregulation depletes NADPH and sensitizes cells to ferroptosis. Conversely, MESH1 depletion rescues ferroptosis by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. Importantly, the ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. MESH1 depletion also triggers extensive transcriptional changes that are distinct from the canonical integrated stress response, but show striking similarity to the bacterial stringent response. MESH1 depletion also leads to dNTP depletion and inhibition of cell proliferation in tumor cells. Finally, we established Mesh1 knockout mouse model to study the physiological relevance of MESH1.

A blood draw is one of the most readily accessible, commonly practiced medical procedures with biomarker utility. In particular, transcriptome signatures of blood cells provide valuable insights into the developmental history and adaptations of these circulating cells. The majority of cells within whole blood consist of erythrocytes, or red blood cells (RBCs) that are primarily responsible for the transport of gases throughout the bloodstream. Terminally differentiated and anucleate, RBCs were once thought to lack most RNAs. However, erythrocytes have been recently shown to contain select microRNAs that lend insight into erythrocyte pathophysiology. Erythrocyte RNAs possess the ability to both distinguish between erythroid disease subphenotypes, and provide insight into mechanisms contributing to these differences. We aim to further dissect the events controlling erythroid differentiation and pathophysiology with these readily-accessible genetic materials.

However, the complete repertoire of either small or large erythrocyte RNAs has not been determined. Based on this knowledge gap, my dissertation has two goals: 1) to define the comprehensive erythrocyte transcriptome, and 2) to utilize an in vitro erythroid differentiation model to elucidate the functions of these newly identified erythrocyte RNAs during development.

Using high-throughput sequencing, we show an extensive, diverse repertoire of both small-sized (short, 18-24 nt) and large-sized (long, >200 nt) RNA species in mature erythrocytes. Though many erythrocyte RNAs have known functions in erythroid cells, we describe several RNAs with unknown functions; these RNAs provide a wealth of genetic loci for further inquiry.

Additionally, several newly described, primate-specific RNAs were identified within the miR-144/-451 locus formerly involved in erythroid development. I performed a functional investigation of a previously uncharacterized microRNA, miR-4732-3p, within this locus. My study demonstrates that this microRNA is upregulated during erythroid development, represses SMAD2/4-dependent TGF-β signaling, and promotes proliferation during erythropoiesis. Thus, miR-4732-3p emphasizes the importance of balanced TGF-β signaling during primate erythropoiesis, and represents a key modulator with potential therapeutic utility.

These two studies highlight the advantage of venipuncture to provide a catalog of erythrocyte RNAs, both for an in vitro and in vivo understanding of erythrocyte biology. This in vitro approach functions as a continuous window into the erythrocyte development program, whereas this in vivo approach provides a snapshot of the mature erythrocyte population state in real-time. The integration of these two approaches provides a broad perspective covering the lifetime of erythroid cells.

Malaria, caused by the apicomplexan parasite Plasmodium, is a disease which affects up to 500 million people each year. Historically, malaria infection has been combated both through the control of its vector, the Anopheles mosquito, and use of a variety of drugs, such as quinine (1800s) and chloroquine (1900s). However, with the evolution of resistance to the majority of available anti-malarial drugs, current approaches have settled upon combinatorial therapies. The most effective of these currently are ACTs (Artemisinin Combination Therapies - Artemisinin derivatives combined with a number of other drugs). However reports of Artemisinin resistance are continuing to emerge, suggesting that new approaches and increased understanding of the Plasmodium parasite is required.

Beginning with the complete sequencing of Plasmodium falciparum genome and continuing with comprehensive profiling of both the parasite's proteome and transcriptome, various genomic approaches applied in the study of malaria have led to significant new insights into the underlying biology of this parasite. While these new findings have greatly increased our understanding of genetic regulation within the malaria parasite, they largely have not yet translated into new therapeutic approaches. For this reason, considerable attention has been paid to the study of human genetic disorders which convey resistance to malaria, in the hopes that elucidating the mechanisms behind these resistances might lead to increased understanding of the parasite's biology and thus novel therapeutic approaches.

Sickle cell (HbS) erythrocytes are well known to resist malaria infection. However, the molecular basis of this resistance, long been recognized as multifactorial, contains elements which remain poorly understood. Here we show that the dysregulated erythrocytic microRNA composition, present in both HbAS and HbSS erythrocytes, is a significant determinant of resistance against the malaria parasite Plasmodium falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition of the target mRNA. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may present a novel host defense strategy against complex eukaryotic pathogens. In addition, the formation of these chimeric transcripts even in normal host erythrocytes illustrates a unique parasitic post-transcriptional adaptation to the host-cell environment.

Malaria persists as a global health problem, with 212 million cases and 429,000 deaths worldwide in 2015 alone. It is caused by the apicomplexan parasite Plasmodium, which follows a complex life cycle that consists of multiple stages spanning from the human host to the mosquito vector. Among the Plasmodium parasites causing human malaria, the deadliest species is Plasmodium falciparum. Most P. falciparum parasites follow an asexual cycle in human erythrocytes that is characterized by a tightly synchronized continuous cascade of gene expression, although a small proportion commits to a sexual fate. Parasites committed to the sexual stage develop into male and female gametocytes over 9-12 days, with mature gametocytes being the only form of the parasite transmissible to the mosquito vector.

This commitment to a sexual fate is rare, and little is known about the transcriptional programs related to sexual commitment and mating-type determination. Furthermore, discrete changes that occur in these cells are largely undetectable in traditional bulk-cell analyses. Bulk-cell analyses were used to establish models for synchronous stage-specific transcriptional programming during the asexual intraerythrocytic developmental cycle (IDC) but left little resolved in terms of cellular heterogeneity and cell-fate decisions. Due to these limitations, we developed unbiased single-cell approaches on a microfluidic platform to analyze single parasites during late asexual and sexual stages. This work was divided into two main parts. The first focused on single-cell gene expression in male and female mid-to-late stage gametocytes. We captured 90 single parasites and compared the expression of 91 genes, including 87 gametocyte-specific genes. Our analysis identified distinct gene clusters whose expression associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. RNA fluorescent in situ hybridization (RNA-FISH) validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses.

The second part of this work centered on single-cell RNA sequencing (scRNA-seq) of P. falciparum late asexual and sexual stages. First, we uncovered a large number of previously undefined gametocyte-specific genes. 46 asexual cells were then segregated into three separate clusters based on the differential expression of SERAs, rhoptries, and EXP2 plus transporters. RNA-FISH of cluster-specific genes validated this distinct stage-specific expression during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.