The Pennsylvania State University The Graduate School

CUE THE STRESS: MALARIA PARASITES EMPLOY CANONICAL AND NOVEL STRESS GRANULES AS A DEVELOPMENTAL MECHANISM

A Dissertation in Genetics by Elyse Elaine Muñoz

© 2020 Elyse Elaine Muñoz

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2020

The dissertation of Elyse Elaine Muñoz was reviewed and approved* by the following:

Robert F. Paulson Professor of Veterinary and Biomedical Sciences Chair of Graduate Program in Genetics Dissertation Advisor

Manuel Llinás Professor of Biochemistry and Molecular Biology Chair of Committee

Andrew Patterson Associate Professor of Molecular Toxicology Associate Professor of Biochemistry and Molecular Biology

David Gilmour Professor of Molecular and Cell Biology Graduate Education Co-Director

ii

ABSTRACT Organisms employ multiple molecular mechanisms to respond to environmental stresses to increase their chances of survival. One of the most well-known mechanisms, the formation of stress granules, has been well-characterized in yeast. These stress granules form in response to many different stimuli, including glucose deprivation, amino acid starvation, heat shock, and oxidative stress. Recently, stress granules have been indicated in human neurodegenerative diseases, including Alzheimer’s disease and dementia. As the ability to clear stress granules decreases, proteins with “sticky regions” are left in the cytoplasm, and begin to coacervate, leading to cell-wide dysfunction that, if left unresolved, results in cell death. This is akin to what is seen in prion diseases, wherein the misfolding of certain proteins allows their aggregation, also resulting in cell death. The formation of stress-granule like complexes have been identified in Plasmodium, the causative agent of malaria. This allows the parasite to generate mRNA transcripts when resources are plentiful and retain them throughout the arduous task of transmission. Then, as the parasite reaches its intended location, the parasite can then release these protected transcripts to allow for their translation and action of the encoded proteins. To this end, characterization of a RNA-binding protein known to be a member of stress-granule like complexes found in Plasmodium species was performed. This protein, ALBA4, has an ancient protein lineage, yet is apicomplexan-specific. The absence of this protein was found to have interesting phenotypes in the transmitted forms of the parasites, including dysregulation of transcripts consistent with the phenotypes. As this RNA homeostasis is critical for the parasite life cycle, this protein is an attractive target to interrogate the underlying basic biology of these stress granules in the parasite context. By performing immunoprecipitations of ALBA4, it was revealed it associates not only with canonical stress granules, but also with mRNA degradative machinery and active translation machinery, highlighting the complexity and dynamic nature of these membrane-less organelles. Finally, experimentation reveals ALBA4 functions are life cycle stage-dependent, again underscoring the complexity of the basic biology employed by this parasite.

iii

Taking these observations together, stress granules are a viable target for therapeutic intervention. The native proteins found within Plasmodium stress granules that contain intrinsically disordered regions, including ALBA4, and a deeper understanding of the clearance of these stress granules, will lead to deeper parallels between yeast and neurodegenerative and prion diseases. By drawing these parallels, it is possible to generate a Plasmodium control strategy centered on perturbing stress granules and/or introducing misfolded peptides that mimic prion proteins. By focusing on the transmission events, this strategy can have the greatest effect at curtailing the spread of this deadly parasite.

iv

TABLE OF CONTENTS List of Figures…………………………………………………………………………………………….…………………………….viii List of Tables………………………………………………………………………………..…………………………………………….x Acknowledgments…………………………………………………………………………………………………………………….xi CHAPTER 1. INTRODUCTION………………………………………………………………………………………………………1 Background…………………………………………………………………………………………………………………….1 Membrane-less organelles: the nucleolus and biomolecular condensates……………………..3 Canonical stress granules in eukaryotes………………………………………………………………..……….7 Stress granule dysfunction and neurodegenerative diseases………………………………………..11 Stress granules in Plasmodium species…………………………………………………………………………14 CHAPTER 2. EXPERIMENTAL METHODS TO ELUCIDATE ALBA4 FUNCTION IN PLASMODIUM……20 Experimental animals and parasite lines…..………………………………………………..………………..20 Reverse genetics of Plasmodium yoelii parasites………………………………………………………….20 Live fluorescence microscopy and indirect immunofluorescence assays………….…………..21 Exflagellation assays…………………………………………………………………………………………………….21 Quantification of gametocytemia…………………………………………………………………………………22 Measurements of mosquito infection and sporozoite development…………………………….22 Immunoprecipitations and western blotting…………………………………………………………………23 Schizont collection and preparation……………………………………………………………….…23 Gametocyte collection and preparation……………………………………………………………24 Oocyst sporozoite collection and preparation…………………………………………………..25 Immunoprecipitations………………………………………………………………………………………25 Western blotting………………………………………………………………………………………………26 Mass spectroscopy……………………………………………………………………………………………………….26 Protein identification……………………………………………………………………………………………………28 Total RNA-seq………………………………………………………………………………………………………………29 Parasite preparation…………………………………………………………………………………………29 RNA preparation……………………………………………………………………………………………….29 RNA-seq……………………………………………………………………………………………………………30

v

Transcript identification and abundance determination……………………………………30 CHAPTER 3. THE ROLE OF TRANSLATIONAL REPRESSION IN SEXUAL AND TRANSMITTED FORMS OF RODENT MALARIA PARASITES…………………………………………………………………………………………….32 Background………………………………………………………………………………………………………………….32 Results………………………………………………………………………………………………………………………….36 PyALBA4 knockout parasites exhibit defects in gametocytes and sporozoites….36 PyALBA4 expression in transmitted forms is consistent with a RNA-granule…….40 PyALBA4 associates with many canonical stress granule components in gametocytes………………………………………………………………………………………….43 PyALBA4 interacts with proteins related to its gametocyte phenotype…………….49 PyALBA4 associated proteins are post-translationally modified in gametocytes………………………………………………………………………………………….50 PyALBA4 affects translationally repressed transcripts in gametocytes………………51 PyALBA4 associates with PyALBA1 and PyALBA2 in oocyst sporozoites…………….53 PyALBA4 affects transcripts encoding RNA-binding proteins in oocyst sporozoites……………………………………………………………………………………………56 Discussion………………………………………………………………………………………………………………….…59 PyALBA4 is likely associating with a canonical stress granule in gametocytes………………………………………………………………………………………….59 PyALBA4 plays an as-yet defined role in sporozoites…………………………………………61 CHAPTER 4. THE ROLE OF TRANSLATIONAL REPRESSION IN ASEXUAL DEVELOPMENT OF RODENT MALARIA PARASITES………………………………………………………………………………………………….63 Results………………………………………………………………………………………………………………………….63 pyalba4- parasites exhibit no defects in asexual or liver stages…………………………63 PyALBA4 plays a multi-faceted role in blood stage schizonts…………………………….64 Proteins associated with PyALBA4 are post-translationally modified………………..69 PyALBA4 affects transcripts involved in invasion in schizonts……………………………70 Discussion…………………………………………………………………………………………………………………….73 PyALBA4 is involved in multiple processes regulating mRNA homeostasis in

vi

schizonts……………………………………………………………………………………………….73 CHAPTER 5. CONCLUSION AND FUTURE STUDIES…………………………………………………………………….78 Conclusions on PyALBA4………………………………………………………………………………………………78 PyALBA4 plays a multi-faceted role in mRNA regulation……………………………………78 PyALBA4 regulates transcripts stage-specifically and stage-independently……….78 Concluding remarks………………………………………………………………………………………….79 Future studies………………………………………………………………………………………………………………80 APPENDIX A – Revisions to Chapter 3…………………………………………………………………………………..….84 APPENDIX B – Contribution to “PlasmoSEP: Predicting surface-exposed proteins on the malaria parasite using semisupervised self-training and expert-annotated data”……………………..87 APPENDIX C – PUF2 KO Analysis……………………………………………………………………………………………….88 APPENDIX D – PUF2 Proposal, ASM Fellowship Proposal…………………………………………….……………89 APPENDIX E – Contribution to “Perk Gene Dosage Regulates Glucose Homeostasis by Modulating Pancreatic β-Cell Functions”………………………………………………………………………………….96 BIBLIOGRAPHY…………………………………………………………………………………………………………………………98

vii

LIST OF FIGURES Figure 1. The nucleolus has three distinct phases: the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC)…………………………………………………………..5 Figure 2. Plasmodium life cycle………………………………………………………………………………………………..15 Figure 3. Trypanosome life cycle………………………………………………………………………………………………33 Figure 4. pyalba4- clonal populations were generated by double homologous crossover recombination and confirmed by genotyping PCR………………………………………………………..36 Figure 5. pyalba4- parasites are adversely affected in gametocyte and sporozoite stages………..37 Figure 6. Scoring key for Plasmodium yoelii gametocytes…………………………………………………………38 Figure 7. pyalba4- parasites do not exhibit significant changes in the number of oocysts per infected mosquito………………………………………………………………………………………………………..39 Figure 8. The total number of sporozoites per infected mosquito remains unchanged in pyalba4- parasites………………………………………………………………………………………………………………………40 Figure 9. PyALBA4::GFP parasites were generated by double homologous crossover recombination and confirmed by genotyping PCR………………………………………………………..40 Figure 10. PyALBA4 is expressed throughout the life cycle and is found in multiple subcellular locations………………………………………………………………………………………………………………………42 Figure 11. PyALBA4::GFP is expressed throughout mosquito stage development, and exhibits two distinct expression patterns in oocysts………………………………………………………………….42 Figure 12. PyALBA4::GFP associates with translational repression machinery and active translational machinery in a largely asexual population, as assessed by immunoprecipitation and nano LC/MS/MS………………………………………………………………….44 Figure 13. PyALBA4::GFP associates with multiple complexes in a stage specific manner, with differing associations in gametocytes and schizonts…………………………………………………….46 Figure 14. Comparison of pyalba4- transcript abundance changes with related published datasets……………………………………………………………………………………………………………………….53 Figure 15. Immunoprecipitation from oocyst sporozoites is feasible with purification and affinity-based techniques……………………………………………………………………………………………..55 Figure 16. PyALBA4 affects transcript levels in both stage-specific and stage-independent

viii

manners……………………………………………………………………………………………………………………….57 Figure 17. pyalba4- parasites do not exhibit any defect in blood stage growth kinetics…………...63 Figure 18. PyALBA4::GFP is expressed throughout mid- to very late liver stages and appears to be packaged into daughter merozoites…………………………………………………………………………64 Figure 19. Model of PyALBA4 roles in asexual and transmitted forms……………………………………..69 Figure 20. pypuf2- clones exhibit a trended decrease in exflagellation…………………………………….88

ix

LIST OF TABLES Table 1. Gametocyte comparison between lines……………………………………………………………………..39

Table 2. pyalba4- parasites show no defect in prevalence of mosquito infection………..……………39

Table 3. pyalba4- parasites show no defect in time to blood stage patency……………………………..40 Table 4. PyALBA4 protein interactions of note in gametocytes………………………………………………..45 Table 5. Full list of detected interactions with PyALBA4 in gametocytes sorted by AvgP.* Table 6. Transcript abundance decreases in pyalba4- gametocytes.* Table 7.Comparison of PyALBA4 interactions with PbDOZI and PbCITH interactions in gametocytes* Table 8. Phosphorylation detected in IP/MS experiments………………………………………………………51 Table 9. Transcript abundance increases in pyalba4- gametocyte*

Table 10. Comparison between the DOZI and CITH RIP-ChIP targets and pyalba4- transcript abundance changes* Table 11. Transcript abundance changes in pyalba4- oocyst-sporozoites*

Table 12. GO term analysis of transcript abundance increases in pyalba4- oocyst-sporozoites……………………………………………………………………………………………………….58

Table 13. GO term analysis of transcript abundance decreases in pyalba4- oocyst-sporozoites……………………………………………………………………………………………………….58

Table 14. Transcript abundance changes in pyalba4- schizonts* Table 15. Full list of detected interactions with PyALBA4 in schizonts* Table 16. PyALBA4 Interactions of Note in Schizonts……………………………………………………………….66 Table 17. Distribution of PyALBA4::GFP localization patterns…………………………………………………..64 Table 18. Comparison between PyALBA4 schizont and PfCK2beta Interactions*

Table 19. GO term analysis of transcript abundance increases in pyalba4- schizonts……………….71 Table 20. Comparison of transcripts with abundance changes across stages* Table 21. Oligonucleotides used in this study…………………………………………………………………………..85 Table 22. Revised oocyst sporozoite table……………………………………………………………………………….86

x

ACKNOWLEDGMENTS There are many, many people who played pivotal roles during my PhD, including family, friends, colleagues, and administrators. Please know that without all of you, I would have mastered out a long time ago! Thank you for everything you have done for me – especially grabbing those late-night beers. There are a few people I would like to mention by name for their outstanding contributions to this work. First and foremost, I would like to thank my parents, Morris and Janelle Muñoz, who taught me it is never too late to go after what you want in life. They taught me to keep my head up, to persevere through any and every obstacle, and to fight for what I believe is just and fair. Without their unconditional support and love, I would not be writing this dissertation. I would like to thank the Huck Institutes, specifically the Genetics program. The admissions committee looked past my less than stellar grades, and focused on my passion and drive for science. I hope I did not let them down. The Huck offered me many opportunities to grow, both personally and professionally, which I did my best to take advantage of. Most importantly, the Huck stood by me and my accomplishments when my character and integrity were called into question. For that, I will always be thankful. Along with thanking the Genetics program, I would be remiss if I did not mention Bob Paulson. Bob, thank you for being the best advisor I could have asked for – even though it took me a long time to realize it. Thank you for guiding me throughout my PhD, and for your unwavering belief in me as a scientist. Without you, this would not be possible – on many levels. Thank you. As many PhDs know, this is one of the hardest things many of us will ever do. It is grueling and can crush your spirit. Without the help of Audra Hixson in the Center for Women Students, I would have left a long time ago. Thank you for helping me see the light, and for the dark reminder that I am not alone and my situation is not unique. Please keep fighting to change that. Finally, there is one last person I would like to thank. Noah Halpert, my partner in life, my husband, my confidant, and my best friend. You have seen me through every up and down

xi over the last six years, and for some reason, you are still here. Your unconditional love and support have held me up when I did not have the strength to stand, and your promise to punch any obstacle in the face always made me laugh and kept me going. Most people do not find the insect vivarium to be a particularly romantic place, but our late dinners and laughing sessions there kept me going when I surely thought I was going insane. Thank you for believing in me when I didn’t believe in myself, and thank you for reminding me the growing pains in this chapter of my life will soon be fading memories. The work included in the following dissertation was conceived, discussed, and approved by Elyse Muñoz, Dr. Scott Lindner, Dr. Manuel Llinás, and Dr. Bob Paulson. Unless otherwise specifically noted, experimentation was carried out by Elyse Muñoz, with support from Lindner lab members. Extensive discussions on data interpretation and analysis were facilitated by Michael Walker, Dr. Heather Painter, and Dr. Joana Santos. Work performed in this dissertation was funded by the National Institutes of Health RO1 DK080040 and USDA-NIFA Hatch Project number 4581 Accession number 1005468, funding from the American Society of Microbiology, and the Alfred P. Sloan Foundation.

xii

For Uncle Mark, the first doctor

CHAPTER 1 INTRODUCTION Background Organisms employ multiple molecular mechanisms to respond to environmental stresses to increase their chances of survival. One of the most well-known mechanisms employed to survive stress is the formation of stress granules. These membrane-less organelles have been well-characterized in yeast. These stress granules form in response to many different stimuli, including glucose deprivation, amino acid starvation, heat shock, and oxidative stress. Interestingly, research has illuminated these stress granules are stress-specific, as different stimuli lead to different stress granule compositions. Recently, stress granules have been indicated in human neurodegenerative diseases, including Alzheimer’s disease and dementia. As neurons age, their ability to clear stress granules lessens. Some proteins found in stress granules contain intrinsically disordered regions, referred to as “sticky regions”. As the ability to clear stress granules decreases, proteins containing sticky regions are left in the cytoplasm and begin to coacervate. This leads to cell-wide dysfunction, and, if left unresolved, results in cell death. Interestingly, this is akin to what is seen in prion diseases, wherein the misfolding of certain proteins allows for their aggregation, also ultimately resulting in cell death. Prion diseases are known to be more debilitating than other human neurodegenerative diseases, potentially due to the rapid misfolding of native proteins. Stress granule formation is driven in part by the liquid-liquid phase separation of components, creating a dilute outer shell and a concentrated inner core of molecules. The underlying biophysics in this phenomenon are the same as those seen in the nucleolus, the largest membrane-less organelle. The nucleolus is the site of ribosome biogenesis and often the site of the first step in a stress-response cascade. In greater eukaryotes, the nucleolus size scales with the amount of translation happening or required for a specific life cycle stage. One reason the nucleolus can rapidly meet the acute translational requirements of a cell with any given stimuli is the liquid-liquid phase separation of its components. Eukaryotic nucleoli are comprised of three separate liquid phases: the fibrillar center (FC), the dense fibrillar

1 component (DFC), and the granular component (GC). Molecules are able to quickly translocate between these different phases, whose formation is composition-driven. The interactions in ribosome biogenesis and phase formation are explored as a standard framework for formation of membrane-less organelles, as well as a critical point of interest in Plasmodium physiology. Recently, the formation of stress-granule like complexes have been identified in Plasmodium, the causative agent of malaria. Despite major advances in understanding of disease etiology, treatments, and vaccine deployment, malaria is still considered a global health concern. Therefore, research to illuminate targetable mechanisms of the parasite when it is most vulnerable – at transmission points between the host and vector – has been a major focus. Several groups have shown membrane-less organelles, or stress-granule like complexes, are vital for proper transmission of the parasite at both transmission points. These stress granules house mRNA transcripts that encode proteins vital for survival post transmission, protecting them from translation and/or degradation prior to the critical moment of transmission. This allows the parasite to generate these mRNA transcripts when resources are plentiful and retain them throughout the arduous task of transmission. Then, as the parasite reaches its intended location, the parasite can easily and rapidly release these protected transcripts to allow for their translation and action of the encoded proteins. Ultimately, this strategy helps the parasite successfully establish infection in an otherwise hostile situation. At these transmission points, the parasite undergoes major population bottlenecks, making the mechanisms they employ at these points attractive targets for therapeutic intervention. To this end, a RNA-binding protein known to be a member of stress-granule like complexes found in Plasmodium species was characterized: ALBA4. ALBA4 has an ancient protein lineage yet is apicomplexan-specific. Its demonstrated function in other apicomplexans includes chromatin remodeling as well as a role in RNA metabolism, ultimately leading to gene or protein expression changes. Interestingly, this protein has been identified in what are hypothesized to be stress granules in Plasmodium, suggesting ALBA4 plays a functional role in protein expression in parasites. In order to interrogate its function and understand the potential implications of perturbing protein expression, an alba4 knockout parasite line was generated. The absence of ALBA4 was found to

2 have interesting phenotypes in the transmitted forms of the parasites, and its further characterization highlighted a single mRNA-binding protein may have multiple functions centered on ensuring proper mRNA homeostasis. This homeostasis is critical for the parasite life cycle, this protein is an attractive target to interrogate the underlying basic biology of these stress granules in the parasite context. By performing immunoprecipitations of ALBA4, it was revealed that it associates not only with canonical stress granules, but also with mRNA degradative machinery and active translation machinery, highlighting the complexity and dynamic nature of these membrane-less organelles. Additionally, dysregulation of transcripts in its absence is consistent with the phenotypes previously noted. Finally, ALBA4 functions are life cycle stage-dependent, again underscoring the complexity of the basic biology employed by Plasmodium. Taking these observations together, it is plausible to suggest stress granules are a viable target for therapeutic intervention. From this work, several experimental pathways have emerged. By exploiting the native proteins found within Plasmodium stress-granules that contain intrinsically disordered regions, including ALBA4, and in turn understanding the clearance of these stress granules, deeper parallels between yeast, neurodegenerative, and prion diseases may be observed. By drawing these parallels, it is possible to generate a Plasmodium control strategy centered on introducing misfolded proteins and/or peptides that mimic prion proteins, and lead to apoptosis. By focusing on the transmission events, this strategy can have the greatest effect at curtailing the spread of this deadly parasite.

Membrane-less Organelles: The Nucleolus and Biomolecular Condensates The nucleolus is the most obvious and critical membrane-less structure within a single cell. Within the nucleolus, rRNA synthesis, processing, and early pre-ribosome assembly occur. On a basic level, the nucleolus is the most easily visible and prominent membrane-less structure within a cell. In eukaryotes, the nucleolus can account for 20-25% of the total nuclear volume in a typical cell. Interestingly, the size of the nucleolus can rapidly expand and decrease as cell demands fluctuate. As ribosome number is correlated with translational demand, in actively growing and/or proliferating cells, the nucleolus is expectedly larger in those cells [1]. The

3 nucleolus is especially suited to housing ribosome biogenesis, as it can easily facilitate the local concentration of specific components and RNA substrates which in turn promotes a rapid ribosome response. This allows for either the production of or continued disassembly of ribosome units, ultimately affecting cell translation level [1]. The nucleolus has the capacity to adapt to rapid translational demand due to its liquid-liquid phase separated layers, each marked by specific ribosome biosynthesis functions and sub-compartments. In eukaryotes, the nucleolus is comprised of three separate layers, driven by surface tension differences: the fibrillar center (FC), dense fibrillar component (DFC), and the granular component (GC) [2-6]. Within each of these phases, additional membrane-less structures, commonly referred to as biomolecular condensates (BMCs) are found. These BMCs are also found throughout the cytoplasm [7-11]. In the nucleus these BMCs include the nucleolus itself, nuclear speckles, and Cajal bodies, amongst others. In the cytoplasm, they include P-bodies, stress granules, Lewy bodies, germ granules, and centrioles, referred to from here forward as canonical stress granules [1]. The main function of the nucleolus, ribosome biosynthesis, is largely conserved amongst organisms and in eukaryotes is achieved through three RNA polymerases responsible for transcribing four non-coding rRNAs, over 150 small nucleolar RNAs (snoRNAs), 80 ribosomal proteins (r-proteins), and over 200 trans-acting assembly factors (AF) [1]. The genesis of ribosomes is initiated by transcription of rDNA genes by the dedicated RNA polymerase I machinery, resulting in production of 47S pre-rRNA precursor. In turn, this initiates nucleation of the nucleolus around the ribosomal genes within the nucleus. Finally, trans-acting AFs coordinate the processing and folding of the 47S pre-rRNA precursor, and its assembly with r- proteins, which occurs in the cytoplasm. Importantly, a crucial quality control step also occurs prior to ribosomal subunit release into the active pool in the cytoplasm [1]. To properly and efficiently achieve proper ribosome biogenesis, the formation of the nucleolus and nuclear BMCs is driven by low-affinity protein-protein interactions [12]. These interactions are driven by intrinsically disordered regions, also called “sticky regions”, found in proteins, and are marked by low-complexity amino acid sequences which allow for high conformational flexibility [13, 14]. This allows for specific coding or non-coding RNAs scaffolding

4

to attract and retain RNA-binding proteins [15, 16]. The resulting ribonucleoprotein (RNP) complexes are responsible for intermolecular low-affinity transient interactions with essential proteins. Importantly, their spatial proximity and composition allows for molecules within a phase-separated liquid condensate to exchange with the surrounding solution while the condensate stably persists [17]. In humans, more than 80% of all RNA found in a growing cell is related to ribosomal RNA, meaning nucleoli exhibit the highest concentration of active genes in the nucleus. It is currently theorized the high concentration of pre-rRNA transcripts is the greatest driver of nucleolus formation [1, 18]. Upon the arrival of pre-rRNA transcripts, proteins are recruited, which in turn drives separation into distinct phases, ultimately allowing for the optimal pre- rRNA processing, transport, and assembly of pre-ribosomal subunits [1]. The transcription of the rDNA occurs in the FC/DFC interface, with pre-rRNA processing and base modifications, such as methylation and pseudouridylation occurring in the DFC. As the pre-rRNA is further processed, the final cleavage step separates the rRNA precursors into the pre-40S and pre-60S particles occurs in the GC [19]. Just prior to movement to the nucleoplasm, the assembly of the pre-40S and pre-60S subunits occurs in the outer GC (Figure 1). Each subunit is separately exported to the cytoplasm for final processing, including assembly, folding, and quality control testing. It is only then that the final AFs dissociate, conferring maturity of the 40S and 60S subunits [20, 21]. Importantly, the outer GC is also a site of sequestration for misfolded unclear proteins, which accumulate during stress

Figure 1. The nucleolus has three distinct conditions [22]. It is hypothesized the storage of the phases: the fibrillar center (FC), dense fibrillar component (DFC), and granular component misfolded proteins in this specific phase prevents (GC). The FC is organized around rDNA gene irreversible aggregation that might impair nuclear arrays, which remain housed and anchored within the FC. When these rDNA genes are function, as well as allows the offending proteins to be transcriptionally active, they are found at the FC/DFC interphase, allowing their nascent pre- substrates for Hsp70-assisted refolding when stress has RNA transcripts to enter the DFC phase. In the DFC, pre-RNA transcripts are modified, and resolved [1]. finally shuttled to the GC, where pre-ribosomal units are loaded onto the transcripts prior to release into the cytoplasm.

5

Quality control of the ribosomal precursors is carried out once they are carried out to the cytoplasm prior to their release into the pool of active ribosomes [1]. To ensure the pre-40S subunit is properly functional, the GTPase activating center is tested in the cytoplasm. The pre- 40S subunit remains in a complex with several AFs, including some that sterically block access to the mRNA channel and initiator tRNA binding P site. Eukaryotic Translation Initiation Factor 5B (eIF5B) and the Fab7 ATPase enables joining the empty pre-40S subunit to the 60S subunit. If this is successful, then Nob1 endonuclease is recruited to generate mature 3’ end of the 18S rRNA. This allows the release of AFs and dissociation of the two subunits, signaling the 40S subunit is mature and functional [23-25]. Within the cytoplasm, BMC formation is dynamic, wherein supramolecular assemblies locate to specific proteins and/or nucleic acids, whose self-assembly and structure are linked to critical and essential cellular processes [26, 27]. Similar to the nucleolus, BMCs exhibit liquid- like properties, and form via liquid-liquid phase separation of the molecular components [2, 28, 29]. The specific components of the membrane-less bodies and their concentration is what drives phase-phase separation within the cell. Spontaneous demixing of molecules allows the formation of distinct dilute and concentrated phases, which are stable, leading to a discrete liquid phase in the center – a core – and a less concentrated outer liquid shell [30]. The resulting membrane-less component essentially sequesters different components into different phases, which may prevent specific biochemical reactions. The most common example in they cytoplasm is stress granules, which sequester mRNA to prevent translation during stress. Due in part to the concentration of proteins in the nucleolus as well as the ease of shuttling proteins due to the phase-phase separation, the nucleolus is highly responsive to stress. Such stresses include changes in environmental conditions causing metabolic stress, DNA damage, as well as proteotoxic stress induced by ubiquitin-proteasome function inhibition. Especially when rDNA transcription and ribosome assembly are perturbed, the nucleolus is the hub for coordinating the stress response until the stress is resolved. As overall ribosome production is decreased during stress, with the potential for preferential translation, this reduction in ribosomes leads to a cascade of nucleolus-mediated molecular events to return to

6 homeostasis. In instances when homeostasis cannot be maintained, a severe response, such as cell cycle arrest and apoptosis, may be initiated [31, 32]. Recent advances in research and proteomics have revealed nucleolar proteins routinely translocate to the nucleoplasm in response to stress. These stresses include exposure to cytotoxic agents, viral proteins, heat shock, ultraviolet radiation, and DNA damage [33-37]. It is likely the translocation of these proteins occurs at a much lower basal level when no stress is applied, however it is significantly upregulated during these stresses. This movement may include the redistribution of particles, including the release of molecules from the nucleolus to the nucleoplasm. As a result, the rapid and unexpected translocation or redistribution can be considered an indicator of nucleolar stress [38]. In Trypanosomes, another parasitic agent closely related to Plasmodium, research has elucidated some significant differences in nucleolus structure. While the nucleolus remains the site for ribosome biogenesis, the tripartite compartmentalization is not present. Instead, only DFC and GC phases are observed, with a noticeably absent FC compartment. While different from eukaryotes, this arrangement is consistent with other protozoans and yeast, and is still thought to be drive by phase separation [39-42].

Canonical Stress Granules in Eukaryotes Gene expression can be regulated at several different levels, including translation and degradation of transcripts, mechanisms that contribute to RNA homeostasis. To achieve this RNA homeostasis, two major membrane-less granules form: stress granules and processing bodies (P-bodies). Canonical stress granules in eukaryotes, such as Saccharomyces cerevisiae, are formed in response to different stressors, including nutrient deprivation, hypoxia, heat shock, and mitochondrial compromise. Stress granules typically contain similar components, including eIF2α, eIF3, eIF4G, eIF4A, eIF4B, eIF4E, and poly(A)-binding protein [43]. Notably, many of the components have either prion-like or poly-glycine rich “sticky” domains, which are hypothesized to allow these stress granules to form and dissociate as needed [44]. However, the components of stress granules are largely determined by the stressor, meaning those formed in response to glucose deprivation do not necessarily contain all or the same

7 components as those formed as a result from heat shock. Further, messenger ribonucleoproteins (RNPs) in stress granules are stalled in various stages of translation, meaning they contain translation initiation machinery [45,46]. Regardless of the stressor, mRNAs sequestered in the stress granules are not translated until the stress is resolved. Stress granules can also form in the absence of external stress, such as in response to blocks in translation initiation, which results in the elimination of translation initiation factors and overexpressing translation repressors [45]. Similar to stress granules, processing bodies (P-bodies) also house mRNAs that are not being translated. However, P-bodies are seen in the presence and absence of stress, and composition of P-bodies is likely independent of the stressor. P-body composition differs from stress granules, as they contain proteins associated with mRNA degradation and more RNA- binding proteins [47]. Canonical P-body proteins include XRN1, CCR4, DCP1/2, and eIF4E, to name a few [48]. Though distinct component-wise, P-bodies are known to spatially overlap with stress granules in yeast and mammals, and have been observed docking together [49, 50]. This indicates the relationship between these two types of granules is dynamic and interconnected, regardless of their differing functions. Additionally, both P-bodies and stress granules are thought to be dynamic structures that can assemble and disassemble as necessary. To further support this assertion, evidence in yeast reveals mRNAs can move between stress granules, P- bodies and polysomes as stress is applied and relieved and translation is resumed [51,52]. The directionality of mRNA movement between these machineries is still unresolved and appears to depend on the type, intensity, and duration of stress [49]. However, this dynamic process allows rapid return to proper RNA homeostasis and protein translation, which gives the cell the important ability to rapidly right itself. Recently, Jain and colleagues took a deeper look into the structural components of stress granules in yeast and mammals, and discovered three important aspects of these granules [47]. Like the nucleolus, stress granules are composed of two different layers. The first is an inner core, which is structured by protein-protein interactions. These interactions are hypothesized to form a stress-granule core when a concentration threshold of mRNA- containing ribonucleoprotein complexes (mRNPs) in the cytoplasm is reached. The second layer

8 is the outer, dynamic shell-like layer, which is less stable, and thought to be governed by liquid- liquid phase separation through weak interactions. This differs from P-bodies, where most components are mRNA-binding proteins, which suggests mRNA may play a larger role in forming structures. Secondly, they showed the dynamic remodeling of these stress granules is ATP-dependent and can be achieved through several ATPase remodeling complexes. This observation is important, as a potential role of these ATPase complexes is to ensure more proteins are located in the outer shell, which may prevent the formation of a larger, more stable stress granule structure that cannot be easily cleared. Additionally, they identified factors, CCT and minichromosome maintenance (MCM) proteins, involved in either the disassembly or persistence of these stress granules, respectively. Finally, they also conclude their data suggests a single protein is not responsible for forming or even triggering the formation of a stress granule, which has been an elusive research target. Instead, they conclude the mechanisms by which stress granules form is likely complex, multi-molecular, yet conserved. This is critical to the understanding of stress granule formation, as it removes the single-target therapy approach and highlights targeting these structures may require multiple targets or disruption of electrostatic, mRNA, and/or protein-protein interactions [47]. Given Parker and colleagues’ observations about the structure of stress granules, questions about their formation have arisen. It is hypothesized the assembly of stress granules occurs when a pool of free mRNPs exists, and these mRNPs form protein-protein interactions between mRNA-binding proteins. These protein complexes are thought to be the core components of stress granules. The outer dynamic shell is then governed by liquid-liquid phase separation interactions [53]. Some proteins thought to be involved in these core protein- protein interactions include Ataxin-2 (Atx2), TIA1 Cytotoxic Granule Associated RNA Binding Protein (TIA-1), Ras-GTPase-Activating Protein SH3-Domain-Binding Protein 1 and 2 (G3BP1, G3BP2), which are necessary but not sufficient to drive stress granule formation [54]. Further, as mentioned previously, these proteins have varying levels of importance in stress granule formation depending on thestressor - G3BP1/2 are important in oxidative stress, but not in osmotic stress [55]. These proteins contain either intrinsically disordered regions (IDRs), prion- like domains (PrLDs), or poly-glycine rich regions. It is hotly contested whether these particular

9 regions are necessary for stress granule formation, especially considering the role untranslated mRNAs play in stress granule assembly [55]. However, a study done by Gilks, et al., showed stress granule formation defects observed in the absence of TIA-1 can be overcome by introducing the PrLD of the yeast protein Sup35 [56]. Additionally, other IDR containing proteins, including Ddx4, LAF-1, and FUS are known to undergo liquid-liquid phase separation in vitro [57-59]. This leads Protter and Parker to suggest IDRs may be important for RNP granule formation, in turn leading to liquid-liquid phase separation of stress granules [53]. However, because of the stable core of stress granules, it is more difficult to reconcile the liquid-liquid phase separation model. Presently, there are two major hypotheses. The first model is stress granule formation is driven by liquid-liquid phase separation, which increases the local concentration of components leading to the core structure [59, 60, 61]. The second model hinges on the stable core formation being independent of IDRs, however, recruitment by the core increases the local concentration of IDRs. This then triggers the liquid-liquid phase separation of the membrane-less organelle. The latter model is preferable, as it is consistent with the findings of a core substructure and a dynamic outer shell and what is seen in the nucleolus. Because protein-protein interactions are critical for the assembly and disassembly of stress granules, questions about post-translational modification of proteins have been explored. A study on G3BP in yeast found phosphorylated G3BP can no longer multimerize, which is an important step in stress granule formation in response to oxidative stress [59]. In terms of disassembly, phosphorylation of Grb7 and DYRK3-kinase promotes stress granule disassembly [62]. Other important post-translational modifications have been interrogated, including methylation and acetylation. Some stress granule proteins contain a RGG motif, which is an important site for arginine methylation. This methylation can be critical for recruitment of proteins to the stress granule, as seen with TDRD3. Point mutations in the Tudor domain of this protein impair its ability to bind methylated arginine and thus its recruitment to stress granules [63]. Acetylation events have importance in stress granules, as HDAC6 is found to be a ubiquitous component in stress granules [64].

10

The final aspect of relevant stress granule biology is their disassembly. Jain et al. showed ATP depletion in in yeast and mammalian cells leads to little to no movement of stress granules [47]. While this is an interesting finding, it is seemingly obvious – many functions within a cell cease in the absence of ATP. Protter and Parker hypothesize the rather stable interactions in stress granules are disrupted by ATPases, and this activity leads to their disassembly once stress is relieved. They further hypothesize the residual material is then cleared by autophagy [53]. To support these hypotheses, they cite work on DEAD-BOX helicases, which are known to unwind DNA and RNA and are ATP-dependent. Though initially thought to be an unlikely member in stress granules, as they are cytoplasmic, studies have shown Ded1 (DDX3) promotes the assembly of stress granules. Further, when its ability to undergo ATP hydrolysis is impaired, mRNAs remain trapped within stress granules [65]. Jain et al. also found minichromosome maintenance (MCM) and Rvb helicases are important for stress granule persistence. In loss-of- function studies of these helicases, there was an increase in the disassembly of stress granules [47]. These findings are surprising, given these are traditionally thought of as nuclear proteins, and it is surprising ATPases would be involved in opposing aspects of stress granule dis/assembly. In terms of autophagy, it is hypothesized this is also governed by an ATPase. The most notable candidate is cell division cycle 48 (CDC-48), which has a demonstrated role in remodeling stress granules to promote autophagy [66]. Inhibiting CDC-48 action leads to stress granule persistence in yeast, which is consistent with its main ATP-dependent function – to extract proteins from complexes so that they may be cleared by autophagy [67].

Stress Granule Dysfunction and Neurodegenerative Diseases Stress granule components have been indicated in neurodegenerative diseases though the link between the two is still unclear. Many neurodegenerative diseases are marked by the presence of protein aggregations, as seen in patient biopsies. Some of these aggregations include beta amyloid plaques, as seen in Alzheimer’s disease, or neurofibrillary tangles, as seen in Alzheimer’s disease, Parkinson’s disease, and primary age-related tauopathy. While the proteins involved in aggregations are not necessarily directly involved in stress granule

11 formation, there is some interesting interplay between them. Interestingly, these diseases generally present later in life, and aging itself is linked to oxidative stress. This chronic stressor may be the link between these pathologies and stress granules. The following is a discussion of how the granules found in neurons – mRNPs, stress granules, and P-bodies – are implicated in neurodegenerative diseases. As previously discussed, there are three major RNA-containing granules found in neurons: mRNPs, stress granules, and P-bodies. These are not drastically different within the neuronal context. The mRNP serves as a largely transportation granule trafficking mRNAs from the nucleus to the cytoplasm, and in the case of neurons from the soma to dendrites to synapses [68]. As seen in yeast, these mRNPs are dynamic and interact with both stress granules and P-bodies [69]. Stress granules observed in neurons function largely the same, though they may suppress apoptosis by inhibiting the MAPK pathway [70]. As in most stress responses, the sequestration of mRNAs in stress granules allows preferential translation of necessary proteins to resolve the stress at hand [71]. P-bodies also function as previously discussed and are known to recruit mRNA by the multimerization domains found on RNA- binding proteins [68]. Several proteins implicated in these diseases are associated with stress granules, either interacting with prominent stress granule components or co-localizing with stress granules or their components. Some of these proteins include Huntingtin [72], PrP prion protein [73], and transactive response DNA binding protein 43 kDa (TDP-43), which forms aggregates seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia [74]. Another noteworthy protein, Tau, can form inclusions within neurons and the presence of these and the resulting neurofibrillary tangles are seen in Parkinsonism-17 and Alzheimer’s disease [75]. Mutations in several proteins important in stress granules are also implicated in neurodegenerative diseases, including fragile X mental retardation 1 protein (FMRP), which results in Fragile X syndrome. Mutations in Ataxin genes result in spinocerebellar ataxia, and in ALS, TDP-43, RNA-binding protein Fused in Sarcoma (FUS), Ataxin-2, optineurin (OPT), and angiogenin (ANG) mutations are associated with the pathology of protein aggregation [76, 77]. The strongest links between protein aggregation and stress granules in these diseases

12 are TDP-43 and Tau. In ALS, the cytoplasmic TDP-43 inclusions are shown to co-localize with stress granule components, specifically TIA-1 and eIF3 [74, 70, 78]. In normal neuronal function, TDP-43 is important for the regulation of splicing mRNAs and maintaining proper mRNA stability [79]. This places TDP-43 within the spatiality of stress granules. Additionally, it is known to contribute to stress granule assembly and maintenance in the context of oxidative stress. It does so by regulating the mRNA levels of G3BP, which as previously discussed is a core component of stress granules [80]. In tauopathic diseases, Tau, a microtubule-associated protein, becomes hyperphosphorylated, resulting in neurofibrillary tangles. Additionally, beta amyloid plaques are also seen in Alzheimer’s disease. Though the two aggregations are distinct, it is possible they influence each other. Beta amyloid plaques form when a certain portion of the amyloid precursor protein misfolds and form aggregates. When this occurs, it may cause a chain reaction, similar to a prion disease. This chain reaction may even trigger Tau to misfold, increasing Tau inclusions [81, 82]. However, this latter interplay is still unclear. While interesting, amyloid beta plaques and Tau inclusion interactions themselves are not tied to stress granules. However, TIA-1 and G3BP accumulate and can co-localize with tau epitopes [79]. An even more obvious link is the amount of TIA-1 positive aggregation rivals the amount of neurofibrillary tangles seen [83]. Evidence for TIA-1 and Tau directly interacting exists; stress stimulates tau phosphorylation, which causes mislocalization of Tau to the soma and dendrites where it interacts with RNA-binding proteins and stress granules [84]. Interestingly, Tau aggregation can be triggered in vitro by the addition of RNA [74]. Taking these observations together, it is hypothesized stress leads to the aggregation of Tau, which in turn stimulates stress granule formation; an increase in stress granules may act in a positive feedback loop and lead to more Tau aggregation. Other than stress granule formation, stress granule clearance may also be affected in these diseases. Mutations in proteins involved in autophagy, such as OPT, ubiquilin-2, DNAJb6, and p62, are also seen in neurodegenerative diseases [85-87]. Taken together, these observations of stress granule formation, association, and clearance within a disease state are all consistent with stress granule persistence, possibly leading to whole cell dysfunction. From

13 these observations, a hypothesis emerges: the persistence of stress granules leads to an increased probability of PrLDs on stress granules interacting and forming a hyperstable beta amyloid-like fiber [88, 61, 89]. This would undoubtedly lead to RNA homeostasis disruption, unnecessary activation of pathways, and/or defects in axonal or nuclear-cytoplasmic transport of mRNPs [90-93].

Stress Granules in Plasmodium Species Malaria remains a major global health threat, with approximately 228 million cases in 2018 alone [94]. The parasite responsible for this disease, Plasmodium species, has a complex life cycle, involving both a host and a mosquito vector (Figure 2). As an infected female mosquito takes a blood meal, sporozoites are injected into the skin of the host. These sporozoites must make their way to the liver, where there develop for approximately two weeks in humans. Once they have fully developed, liver cells are lysed and the parasites find their way into the bloodstream. At this point, a single parasite will invade a single blood cell. The parasite has three separate blood stages: rings, trophozoites, and schizonts. As the parasite transitions to a schizont, it undergoes asexual replication. After their maturation is complete, the red blood cell is lysed and the parasites are released to find a new red blood cell. A small subset of these parasites will undergo a different developmental pathway and become sexual forms – the gametocytes. It is this form of the parasite that results in successful infection of the female mosquito when it takes a blood meal from an infected host. Within the gut of the mosquito, the male and female gametocytes fuse to form a zygote, rapidly developing into the motile ookinete. The parasite then burrows through the midgut of the mosquito and takes residence to form a cyst. Each of these cysts forms thousands of sporozoites, which travel to the salivary gland of the mosquito and wait to be injected in their next host. The transmission of the parasite, both from vector-to-host and host-to-vector, has been a major focus of research due to the parasite population bottlenecks that occur at these points with hopes of exploiting this weakness [95]. The transmitted stages, gametocytes (host-to-vector) and sporozoites (vector-to-host) have been effectively targeted by drugs and vaccine candidates [96, 97]. Of these two stages, gametocytes have been studied on a deeper biochemical level as

14 they have proved more experimentally tractable, with easier production and purification in large numbers possible in a lab setting. Studies of gametocytes have led the field to appreciate the essentiality of translational repression in successful infection and parasite propagation [97- 99]. However, it is still unclear whether this translational repression is achieved through bona fide stress granules or a similar RNA storage granule. Given the complex life cycle of Plasmodium, there are several points were significant stress are applied to the parasite and may trigger the formation of stress granules. For example, as the parasite is transmitted from the host to the vector, the parasite is exposed to a rapid drop in temperature, different pH environment Figure 2. Plasmodium life cycle in the mosquito, and potentially a different oxidative environment. As the parasite develops in the gut of the mosquito, utilizing metabolic resources of the vector, resource deprivation may occur, which can also trigger a stress response. As the parasite is transmitted from the vector to the host, the parasite is again exposed to rapidly changing temperature, pH environment, and availability of resources. Nutrient deprivation seems less likely, given the vast amount of resources available in the mammalian host. However, another stressor may be assault with anti-malarials, as patients seek and receive treatment. Though classic stressors have not been applied to study the formation of stress granules and/or P-bodies in Plasmodium species, there is evidence of their formation in response to stress. Oakley et al. [100] demonstrate in response to increased temperature, Plasmodium parasites shift RNA homeostasis, down-regulating translation initiation factors and increasing stress granule markers and RNA-binding proteins. In addition, Oakley also explored the effects of radiation used to generate attenuated parasites for vaccine purposes and found while translation initiation factors were down-regulated, genes associated with mRNA stress granules

15 and RNA-binding proteins were upregulated [101, 102]. These observations indicate stress granules may form in these parasites. In Plasmodium, RNA-binding proteins are implicated in translational repression; RNA-binding proteins, such as DOZI and CITH, show increased protein expression during gametocyte and sporozoite stages when certain transcript abundances are high but their respective protein is near absent. Hall et al. [103] provided the first comprehensive and comparative look across Plasmodium genomes, transcriptomes, and proteomes. Their analyses confirmed the findings of Paton et al. [104], which showed though transcript abundances of Pbs21 were high in female gametocytes, protein expression was near absent. However, the protein is expressed after activation of the gametocyte to form a gamete. This provides evidence for translational repression as a mechanism for gene expression regulation. In Plasmodium, both transcriptional and translational repression have been characterized as crucial processes for proper development and transmission. In rodent- infectious Plasmodium berghei, the specific transcription factor AP2-G2 was recently found to be responsible for the transcriptional repression of asexual development factors, and thus promotes conversion to gametocytes [105]. Additionally, the parasite uses RNA storage granules to store and translationally repress specific mRNAs during transmission [106, 107]. Studies of the gametocyte and sporozoite stages demonstrate while transcripts are present, the proteins they encode are absent or in extremely low abundance. Comparing recent total transcriptomic and proteomic datasets, the difference is especially striking for some of the most abundant transcripts in the gametocyte (e.g., p25 and p28) and the sporozoite (e.g., UIS3 and UIS4). These particular transcripts show evidence of very strong translational repression, as other transcripts that are equally abundant (e.g. CSP, TRAP) exhibit far more protein [103, 106, 108]. This specific translational repression is relieved following transmission, as the encoded proteins are expressed after activation of the gametocytes to form a gamete and following hepatocyte invasion by a sporozoite [109]. This translational repression and derepression are critical to the parasite post-transmission, as these proteins are essential for parasite developmental program to initiate at these points [110-112]. Work to understand the mechanisms employed to impose specific translational

16 repression upon select transcripts during Plasmodium transmission has focused on a few RNA- binding proteins: DOZI, CITH, Puf2 and the ALBA protein family [106, 107, 113-118]. As transmission studies are more easily conducted with rodent-infectious species, we and others have used P. yoelii and P. berghei species to show these proteins are critical to the transmitted forms of the parasite [119, 120]. In sporozoites, Puf2 is crucial for maintaining the infectivity of sporozoites while residing in the salivary gland of mosquitoes and disrupting Puf2 genetically leads to premature dedifferentiation into a liver stage-like form while still inside the mosquito [98, 106]. Members of the Puf protein family, named after their discovery in Drosophilia (Pumilio) and C. elegans (FBF), are characterized by the presence of eight Puf repeats near the C-terminal end of the protein which form the RNA-binding domain [121]. In Drosophila, Pumilio acts as a translational repressor, inhibiting the translation of hunchback mRNA to allow for anterior/posterior patterning of the embryo [122]. Serving a similar function in C. elegans, FBF represses fem-3, which allows the switch from spermatogenesis to oogenesis to occur [123]. Puf proteins are widespread and found throughout eukaryotes, including yeast and vertebrates, as well as Plasmodium and other protozoan parasites, such as Trypanosomes, Leishmania, and Toxoplasma species [124-126]. In Plasmodium, two Puf proteins (Puf1, Puf2) have been identified and characterized in several species, including the human-infectious P. falciparum and rodent-infectious P. berghei and P. yoelii. Miao et al. [127] showed PfPuf2, though not essential, plays a critical role in gametocytes; in the absence of Puf2, gametocytemia was increased and premature differentiation of the male gametocytes was seen. In P. yoelii, Lindner et al. [106] showed Puf2 was crucial for maintaining infectivity of sporozoites while residing in the salivary gland of mosquitoes. Further, Lindner demonstrated and confirmed Puf2 forms novel RNA granules that resemble, but are distinct from, stress granules and contribute to RNA Homeostasis, presumably by translationally repressing certain transcripts that are necessary for liver infection [115]. These stress granules do not co-localize with Plasmodium-specific eIF2α, a canonical stress granule component in eukaryotes, or with Plasmodium-specific XRN-1 or SAP1, canonical degradation machinery components found in P-bodies. Yet, these granules are similar in size, location, and distribution to stress granules and P-bodies. Excitingly, recent work shows

17

Puf2 binds to the transcripts of both p25 and p28 in certain life cycle stages, and translationally represses them [128]. These transcripts are known to be highly expressed in female gametocytes yet have little to no protein present at this life cycle stage. the proteins encoded by these transcripts are found downstream developmentally, as the female gametocyte fuses with a male gametocyte in the mosquito. It is important to note there is a paucity of classic stress granule immunohistochemistry markers available in Plasmodium, though homologs for the genes are present and predicted to perform similar conserved functions. Therefore, there may be more overlap between these novel storage granules and canonical stress granules than demonstrated in the work described above. In support of the novel storage granule theory, however, is the demonstration by Lindner and colleagues that Puf2 granules do not co-localize with eIF2α protein, a canonical eukaryotic stress granule component, in sporozoites [106]. It is currently unknown what other proteins and/or RNAs associate with Puf2 to form these RNA granules. Similarly, other RNA-binding proteins that associate with canonical stress granules are expressed during key transmission points in the Plasmodium life cycle. The orthologue of the DDX6 DEAD-box RNA helicase (called DOZI) is a component of cytoplasmic RNA granules in female gametocytes, and acts as a translational repressor [107]. In the absence of DOZI, P. berghei female gametocytes are unable to produce functional zygotes post fertilization. Without the specific translational repression imposed by DOZI, transcripts of proteins required post-fertilization are prematurely translated, and the parasites are unable to enter into meiosis [107]. Follow-up studies with another RNA-binding protein known to associate with DOZI, called CITH (a homologue of CAR-I and Trailer Hitch), also demonstrated a highly similar phenotype in zygotes [116]. In model eukaryotes, DDX6 and Scd6/Lsm14A (CITH homologs) are present in both stress granules and P-bodies [47]. In Trypanosomes, these proteins are also found in nuclear periphery granules (NPGs). These findings are consistent with their role in translational repression in Plasmodium gametocytes. More recently, work in Trypanosomes has identified NPGs as another type of membrane-less granule, named for their nuclear adjacent localization [129]. Trypanosomes are another protozoan parasite similar to Plasmodium, which also have a complex life cycle

18 including a mammalian host and insect vector. During the procyclic stage of the life cycle, which takes place in the Tsetse fly, Kramer and colleagues determined NPGs form when trans-splicing is inhibited, and thus mRNA maturation is incomplete. Interestingly, NPG protein composition excludes proteins involved in mRNA maturation or export. Instead, the protein components of NPGs almost exclusively overlap with P-bodies, including eIF4E1, CAF1, XRNA, DHH1, and SCD6. Interestingly, eIF4E1 is a translation initiation factor, while CAF1 and XRNA are involved in canonical mRNA degradation. DHH1 and SCD6 are RNA-binding proteins, homologs to Plasmodium’s DOZI and CITH, discussed above. However, poly(A) binding protein 2 (PABP2) and the homolog of the Drosophila protein VASA are also detected [129]. As these proteins are not known to be involved in mRNA degradation or P-bodies, their inclusion in NPGs further demonstrates the highly differentiated and complex granule dynamics at play. Because these proteins are found in NPGs, it is suggested these NPGs are more similar to P granules of C. elegans and nuages of Drosophila [129]. This observation further underscores the complexity of identifying and characterizing granules in these organisms and suggests there are likely other yet-to-be-defined granule types contributing to post-transcriptional regulation in eukaryotes.

19

CHAPTER 2 EXPERIMENTAL METHODS TO ELUCIDATE ALBA4 FUNCTION IN PLASMODIUM

Experimental animals and parasite lines Six to eight week old female Swiss Webster (SW) mice were obtained from Harlan Laboratories (Harland, IN) and were used for all experiments described. Plasmodium yoelii parasites were cycled between these mice and Anopheles stephensi mosquitoes reared at 24°C and 70% humidity. All animal care strictly followed the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines and was approved by the Pennsylvania State University Institutional Animal Care and Use Committee (IACUC# 42678).

Reverse genetics of Plasmodium yoelii parasites

Transgenic parasites (pyalba4-- and PyALBA4::GFPmut2) were generated by producing a gene-targeting construct as previously described [106]. Briefly, two regions of the pyalba4 locus were PCR amplified using Phusion polymerase (NEB) and specific primers (See Table 21 in Appendix A). Resulting products were gel extracted (QIAquick Gel Extraction Kit, Qiagen, Cat# 28706), precipitated with ethanol, and then fused together by Sequence Overlap Extension (SOE) PCR to create the final targeting sequence [156]. This product was also gel extracted and precipitated with ethanol, then inserted into an intermediate vector for sequencing and amplification (pCR-Blunt, Invitrogen), and ultimately subcloned into a modified pDEF vector designed to allow for double crossover recombination events [106]. Plasmids for gene disruption or C-terminal tagging were linearized at a unique restriction enzyme site between the targeting sequences, precipitated by ethanol, dissolved in ddH2O, and then transfected into WT parasites as previously described [157]. Briefly, transfected parasites were selected with pyrimethamine (Fisher Scientific, Cat# ICN19418025) by drug cycling (3 days on, at least 3 days off), and allowed to reach 1% parasitemia. Parasites were then transferred into naive mice followed by another round of pyrimethamine drug cycling. Parasites were harvested and genomic DNA was isolated and purified (QIAamp DNA Blood Kit, Qiagen, Cat# 51106). The presence of the transgenic locus was confirmed by genotyping PCR. Clonal populations of the

20 initial knockout parasites were isolated by limited dilution cloning and the transgenic locus was also confirmed by genotyping PCR. Similarly, a wild-type parasite expressing GFPmut2 from a dispensible locus (p230p) was created and isolated (called WT-GFP throughout). Briefly, the pDEF vector was modified to contain two Py p230p targeting sequences (see Table 21 in Appendix A for primers used) for double crossover recombination and expression cassette with the PyEF1a promoter driving GFPmut2 expression with a PbDHFR 3’UTR appended. Verification of the transgenic locus was conducted by genotyping PCR, and the transgenic population was enriched by FACS to select the brightest parasite population.

Live fluorescence microscopy and indirect immunofluorescence assays (IFA) PyALBA4::GFP expression in blood stages, oocyst sporozoites, salivary gland sporozoites and liver stages was visualized by indirect immunofluorescence assay. For IFAs, all samples were prepared as previously described [145], and stained at RT with the following primary antibodies: rabbit anti-GFP (1:1000, Invitrogen, Cat# A11122), rabbit anti-ACP (1:1000, Pocono Rabbit Farm & Laboratory, Custom PAb), mouse anti-GFP (1:1000, DSHB, Clone 4C9), rabbit anti-DDX6 (1:1000, gift from Joe Reese, Custom PAb), mouse anti-alpha tubulin (B-5-1-2) (1:1000, Sigma, Cat# T5168), and mouse anti-CSP (1:1000, Clone 2F6 [108]). Secondary antibodies used were Alexa Fluor-conjugated (AF488, AF594) and specific to rabbit or mouse (1:1000, Invitrogen, Cat# A11001, A11005, A11008, A11012), and 4ʹ,6-diamidino-2-phenylindole (DAPI) was used to stain nucleic acids. Samples were covered with VectaShield antifade reagent (Vector Laboratories, VWR, Cat# 101098-048) and a coverslip, and sealed with nail polish. For live fluorescent images, mosquito midguts in 1xPBS or sporozoites in Schneider’s medium (Fisher Scientific, Cat# BW04-351Q) were placed on a slide and carefully covered with a coverslip. Fluorescent and DIC images were taken using a Zeiss fluorescence/phase contrast microscope (Zeiss Axioscope A1 with 8-bit AxioCam ICc1 camera) using a 40X or 100X oil objective and processed by Zen imaging software.

Exflagellation assays Mice were infected with either WT-GFP or one of two independent clones of the

21 pyalba4- parasite line (pyalba4- Clone 1 or pyalba4- Clone 2). Two mice were infected per parasite line per biological replicate and infections were monitored daily in peripheral blood by tail snip. The number of exflagellation events was determined daily 10-15 minutes after blood collection by counting the number of centers of movement in ten 40x phase contrast fields on a monolayer of blood cells. Six biological replicates were completed with all clones. For pypuf2- parasites, a single mouse was infected per parasite line (WT-GFP, two independent pypuf2- clones) per biological replicate, and allowed to reach 0.5-1% parasitemia. Parasites were harvested by cardiac puncture and approximately 1000 mixed blood stage parasites from each line in 100uL RPMI (Corning, VWR, Cat# 45000-412) were injected by tail vein into two naive mice per parasite line per biological replicate. The number of centers of movement was monitored daily in peripheral blood by tail snip as mentioned above. Two biological replicates were completed with all clones.

Quantification of gametocytemia

Thin blood smears were taken from mice infected with WT-GFP, pyalba4- Clone 1 or pyalba4- Clone 2 parasites on the days of peak number of centers of movement. Smears were fixed with methanol and stained with a buffered 6.7mM Giemsa (Sigma, Cat# GS80) solution

(417mM Na2HPO4, 227mM Na2HPO4H2O, pH 7.2) for 7-10 minutes. Smears were viewed under 100X oil objective and the numbers of mature female, mature male, and immature gametocytes were counted per ~10,000 red blood cells (See Figure 6 for a key for these gametocyte populations). The fields selected for viewing followed the St. Andrew’s cross pattern across the smear, as gametocytes tend to collect at the leading edge of the smear [158].

Measurements of mosquito infection and sporozoite development

Mosquitoes infected with either WT-GFP, pyalba4- Clone 1, pyalba4- Clone 2 parasites were dissected at Days 5, 7, 10, 12, 14, 16, 18, and 20 post-blood meal to assess aspects of these infections. To assess prevalence of infection (percentage of mosquitoes infected), 10-20 mosquitoes from D5-D10 were dissected under a stereomicroscope in Schenider’s medium.

22

Midguts were placed in 1X PBS on a slide and covered with a coverslip, then visualized with fluorescence microscopy. A mosquito was considered infected if at least one oocyst was visible in the midgut. The number of oocysts per midgut was similarly quantified by fluorescence microscopy. To assess sporozoite development, 10-25 mosquitoes infected with each parasite line were dissected on D10-D20. Both the midgut and the salivary glands were isolated from the same mosquito and placed into separate microfuge tubes. Each was ground with a pestle to release the sporozoites. The tubes were spun at 900 rpm for 30 seconds in a tabletop centrifuge, and the supernatant containing the released sporozoites was collected with a glass Pasteur pipette. Sporozoites were diluted in Schneider’s medium and loaded onto a Hausser Bright-Line Phase hemocytometer (Fisher Scientific, Cat# 02-671-6) and quantified by phase contrast microscopy. Counts were adjusted for prevalence of infection and are reported as parasites per infected mosquito. Three biological replicates were completed with all clones. Mosquitoes infected with PyALBA4::GFP parasites were assessed for prevalence on D5- D10 as above. On D10 and D14 midguts and salivary glands were isolated, respectively, and sporozoites were released and quantified as mentioned above.

Immunoprecipitations and western blotting Asexual (schizont) collection and preparation To collect an enriched schizont population, SW mice were infected with WT-GFP or ALBA4::GFP (4 mice per parasite line) and allowed to reach 1-2% parasitemia (assessed by Giemsa staining). Mice were sacrificed and the infected blood was removed by cardiac puncture exsanguination, washed in media (20% heat inactivated fetal bovine serum (Fisher Scientific, Cat# MT35016CV), 80% RPMI (Corning, VWR, Cat# 45000-412), 0.028% Gentamicin) to remove mouse serum, and then incubated in media and blood gas mixture (10% O2, 5% CO2,

85% N2) for ~12 hours on an orbital shaker (50 rpm) at 37°C. Cultures were transferred to 50mL conical tubes and a discontinuous gradient (composed of 60% stock Accudenz solution (27.6% w/v Accudenz (VWR, Cat# 100328-260) in 5mM Tris-HCl (pH7.5@RT), 3mM KCl, 0.3mM EDTA, filter-sterilized) and 40% commercial 1X PBS (VWR, Cat# 45000-446) was carefully laid under the blood culture. Cultures were spun at 200g for 20 minutes at RT with low acceleration and

23 no brake, and mature schizonts and gametocytes were collected from the interface with a glass Pasteur pipette. Parasites were spun again at 200g for 10 minutes at RT with low acceleration and with no brake into a loose pellet. Parasites were immediately fixed following the above- mentioned spin with a 1% formaldehyde (VWR, Cat# PI28908) in 1X filtered-PBS solution or RPMI media (80%RPMI, 20%FBS) for 10 minutes at RT. The fixation was quenched by the addition of 1/10 volume of1.25M glycine in ddH2O for 10 minutes, spun for 5 minutes at 9,200g at RT, and then stored at -80°C until time of use.

Gametocyte collection and preparation Parasites were purified, lysed and incubated with Dynabeads MyOne Streptavidin T1 (Life Technologies, Cat#65601) coated with a biotin-conjugated GFP antibody (Abcam, Cat# ab6658). In detail, to collect an enriched gametocyte population, SW mice were infected with WT-GFP or ALBA4::GFP (4 mice per parasite line) and allowed to reach 1-2% parasitemia (assessed by Giemsa staining). Mice were then treated with a 10mg/L sulfadiazine solution for two days, or until asexual stages were no longer seen by thin blood smear. Mice were exsanguinated by cardiac puncture, and the blood was placed directly into heated media (37°C; 20% heat inactivated fetal bovine serum (Fisher Scientific, Cat# MT35016CV), 80% RPMI (Corning, VWR, Cat# 45000-412)). An Accudenz discontinuous gradient (composed of 60% stock Accudenz solution (27.6% w/v Accudenz (VWR, Cat# 100328-260) in 5mM Tris-HCl (pH7.5@RT), 3mM KCl, 0.3mM EDTA, filter-sterilized) and 40% commercial 1X PBS (VWR, Cat# 45000-446)) was immediately carefully laid under the culture. Cultures were spun at 200g for 20 minutes at RT with low acceleration and no brake, and gametocytes were collected from the interface with a glass Pasteur pipette. Parasites were spun again at 200g for 10 minutes at RT with low acceleration and with no brake into a loose pellet. Male gametocytes remained largely un-activated through this purification, as exflagellation events were not observed following this spin, but were observed in an aliquot of these samples that was subjected to wet mounting on glass slides and incubation at room temperature for 10 minutes (data not shown). Parasites were immediately fixed following the above-mentioned spin with a 1% formaldehyde (VWR, Cat# PI28908) in 1X

24 filtered-PBS solution or RPMI media (80%RPMI, 20%FBS) for 10 minutes at RT. The fixation was quenched by the addition of 1/10 volume of1.25M glycine in ddH2O for 10 minutes, spun for 5 minutes at 9,200g at RT, and then stored at -80°C until time of use.

Oocyst sporozoite collection and preparation Approximately 600 mosquitoes were dissected per parasite line (WT-GFP and PyALBA4::GFP), resulting in 7-10 million oocyst sporozoites. Midguts were ground in Schneider’s medium, and purified once by previously described discontinuous gradient method [143]. Briefly, released sporozoites resuspended in 1mL of Schneider’s medium were loaded onto a 2.5mL 17% w/v Accudenz cushion (17% w/v Accudenz in molecular grade H2O (Fisher Scientific, Cat# SH30538LS)) in a 15mL conical tube (Fisher Scientific, Cat# 14-959-70C), and spun at 2500g for 15 minutes at RT with low acceleration and no brake. Sporozoites were collected from the interface with a glass Pasteur pipette and placed in a microfuge tube. Sporozoites were quantified by hemocytometer, pelleted (2 minutes at 9,200g at RT), and placed in -80°C until time of use.

Immunoprecipitation Fixed parasite pellets were thawed and lysed in RIPA buffer (50 mM Tris-HCl (pH 8.0@RT), 0.1% w/v SDS, 1mM EDTA, 150 mM NaCl, 1% v/v NP40, 0.5% w/v sodium deoxycholate) with a 1x protease inhibitor cocktail (Roche, VWR, Cat# PI88266) and 0.5% SUPERase In (Life Technologies, Cat# AM2694) for 1 hour at 4°C. Notably, sporozoites were not were not fixed or crosslinked. As a precaution, a modified lysis buffer was used (50mM Tris-HCl (pH7.5), 150mM NaCL, 1% v/v NP-40, 1% v/v Triton-X 100, Protease inhibitor cocktail (Roche), 5% glycerol, and SuperRNase). Following lysis, lysates were homogenized by a 1mL Dounce homogenizer (VWR, Cat#62400-595) using the “Tight” pestle. Samples were dounced 10 times each. Samples were then sonicated at 10% duty cycle with four 0.5 second pulses, and then spun to remove insoluble material post-lysis. The lysate was transferred to a new microfuge tube and 5% was set aside as ‘Total Input’. To reduce nonspecific capture, lysates were incubated with blank Dynabeads MyOne Streptavidin T1 (Life Technologies, Cat# 65601)

25 treated for RNA manipulation per manufacturer protocol. Briefly, beads were washed twice in a 0.1M NaOH (VWR, Cat# 97064-526), 0.05M NaCl (Fisher Scientific, Cat# BP358-10) in DEPC- treated H2O (Fisher Scientific, Cat# BP5611) solution. Dynabeads were then washed in a 0.1M

NaCl in DEPC-treated H2O solution, followed by 4 washes with 1X filter-sterilized PBS. Lysates were incubated with these Dynabeads for 1 hour at 4°C with end-over-end rotation. Lysates were then transferred to a microfuge tube containing Dynabeads coated with biotin-conjugated GFP antibody. These Dynabeads were also treated for RNA manipulation as above, then allowed to incubate with the biotin-conjugated antibody in 1X filtered-PBS for 30 minutes at RT with gentle rocking. Newly conjugated Dynabeads were then washed 4 times with 1X filtered- PBS. Lysates were incubated with these Dynabeads for 3 hours at 4°C with end-over-end rotation. Supernatant was retained as ‘Flow Through’, and the Dynabeads were washed four times with a modified-RIPA buffer containing no SDS or sodium deoxycholate. To elute and reverse the crosslinking, the beads were incubated in Sample Buffer (50mM Tris-HCl (pH 6.8@RT), 5% w/v SDS, 5% glycerol, 0.16% Bromophenol Blue, 5% β-mercaptoethanol added just before use) containing 200mM NaCl overnight at 45°C on a digital dry heat block.

Western blotting To confirm the pull-down of GFPmut2 and ALBA4::GFP proteins, a western blot was performed using the Total Input (2.5% of total sample volume), Flow Through (3.5% of total sample volume, and Elution (half of total elution) from the WT-GFP control and the ALBA4::GFP samples. Blots were probed with primary rabbit anti-GFP (1:1000, Invitrogen, Cat# A11122) and secondary rabbit IgG conjugated with horseradish peroxidase (1:5000, Invitrogen, Cat# A16104), then visualized by exposing film to the blot following treatment with SuperSignal West Pico chemiluminescent substrate (VWR, Cat# PI34080).

Mass spectroscopy The elution samples from the immunoprecipitations were prepared for liquid chromatography-mass spectrometry (LC/MS-MS) as follows. Samples were heated to 70˚C for 5 min, then electrophoresed through a Thermo 4-20% gradient gel (Cat# PI25204) at 150V at RT

26 for 30 min. The gel was stained with Imperial Stain (Fisher Scientific, Cat# PI-24615) as per manufacturer’s recommendations for 1.5 hours and then destained for 1.5 hours in commercially prepared dH2O. Sample lanes were cut into 4 equal gel slices, and each of the gel sections were diced into ~ 1 mm3 pieces and washed three times with approximately five gel volumes of a solution consisting of 50% v/v acetonitrile (ACN) and 50mM triethylammonium bicarbonate (TEAB) in dH2O for 20 min at 37˚C. Disulfides were reduced and alkylated by incubating the gels in a solution consisting of 5mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and 50mM TEAB in dH2O for 10 min at 60C followed by a 15 min incubation in a fresh 100mM solution of iodoacetamide in 50mM TEAB at 37˚C with mixing in an Eppendorf Thermomixer R. The reducing and alkylating reagents were then removed by washing the gel slices in 50% ACN, 50mM TEAB in dH2O three times as described above and dehydrated in 100% ACN. Residual ACN was removed in a Speedvac (Thermo). A 1 g/L stock solution of sequencing-grade trypsin (Thermo Cat# 90055) in 1mM hydrochloric acid was diluted 100-fold with chilled 50mM TEAB in dH2O, and the dry gels were allowed to completely re-hydrate in the protease solution. Proteolysis was carried out overnight at 37˚C. The peptides were extracted with three 50L volumes of 50% ACN, 0.1% formic acid (FA) in dH2O and one 50L volume of 100% ACN, dried down, and reconstituted in

10L of 4% ACN, 0.1% FA in dH2O for the LC MS2 analysis. For each sample, 1.5uL (of 10ul total) was loaded onto an Acclaim PepMap100 trapping column (100 m × 2 cm, C18, 5 m, 100 A, Thermo) at a flow rate of 20L/min using 4% aqueous acetonitrile (ACN), 0.1% formic acid (FA) in dH2O as a mobile phase. The peptides were separated on an Acclaim PepMap RSLC column (75 m × 15 cm, C18, 2 m, 100 A, Thermo) with a

90 min 4% - 60% linear gradient of acetonitrile in dH2O containing 0.1% formic acid. The gradient was delivered to the column by a Dionex Ultimate 3000 nano-LC system (Thermo) at 300nL/min.

An LTQ Orbitrap Velos mass spectrometer (Thermo) was set up for a ‘2nd Order Double Play’ type of experiment with the following parameters: full positive-ion 1000-ms FT MS scan at

R 60,000 over 350 – 1700 m/z followed by ten ion-trap MS2 scans on most intense precursors with collision-induced dissociation (CID) activation. Precursor ion signal threshold was set at

27

5000, isolation width 2 m/z, normalized collision energy 35.0 V, activation Q 0.250, activation time 10.0 ms. The precursors were selected using an FT master scan preview mode with charge states less than +2 rejected and monoisotopic precursor selection enabled. Dynamic exclusion repeat duration was 25 s, exclusion duration 13 s, exclusion list size was 200, and the exclusion mass width was +/- 10 ppm relative to the excluded m/z. Polysiloxane signal (m/z 445.1200) was used as a lock mass.

Protein Identification The resulting data was processed using the Trans-Proteomic Pipeline (TPP) [146] essentially as described previously with some modifications [108]. In brief, raw mass spectra were converted to .mzML format using MSConvert [147] and searched by both X!Tandem [148] and Comet [149]. Spectra were searched against reference sequences downloaded on January 21, 2015 for Plasmodium yoelii 17X (from PlasmoDB), mouse (from Uniprot), common contaminants (cRAP-ome, from http://www.thegpm.org/crap/), and decoys generated by TPP. Similar search parameters were used for both X!Tandem and Comet searches as previously described [108]. The MS/MS data were analyzed using TPP (v4.8.0), and peptide spectrum matches (PSMs) produced by X!Tandem and Comet were analyzed independently using Peptide Prophet to assign probabilities of being correct for each PSM. All Peptide Prophet scores for both search types were combined in iProphet, and protein identifications were inferred by Protein Prophet [150, 151]. Only those proteins with a false positive error rate of less than 1.0% are reported. To combine replicate datasets, the SAINT algorithm, version 2.5.0 was used [152]. Briefly, the algorithm normalizes the total spectral abundance for each inferred protein, as well as protein length to calculate the probability of an interaction with the bait (PyALBA4::GFP in this case). The algorithm was run with the following settings: lowMode=1, minFold=0, normalize=1. Only those proteins with a false positive error rate of less than 10% were considered significant hits and included in the analyses, as this threshold has been used before [144]. Bioinformatic searches for post-translational modifications in this targeted set of

28 proteins were performed by producing a new reference database curated to include only sequences of proteins previously inferred below a 1% FDR in each dataset, along with decoy sequences specific to this set. The data was processed using the TPP [146]; database searches were performed by X!Tandem [148]. Separate searches for modifications of +79.96633 Da for phosphorylation of serine, threonine, or tyrosine, or + 42.01056 Da for acetylation of lysine were conducted. The PSMs produced by X!Tandem were analyzed using Peptide Prophet to assign probabilities of being correct for each PSM. Protein identification with modified residues were inferred by Protein Prophet [150, 151]. Only those modified proteins with a false positive error rate of less than 1.0% are reported. The raw and fully analyzed data files for all mass spectrometry-based experiments have been deposited in PRIDE (Accession #PXD004183).

Total RNA-seq Parasite preparation To generate parasite samples, parasites were collected from gametocytes, oocyst sporozoites, or schizonts as mentioned above. Following purification by Accudenz gradient, mouse RBCs were subjected to lysis with 0.1% w/v saponin in 1xPBS, then washed with 1xPBS. Released parasites were then lysed immediately. Oocyst sporozoites were produced in An. stephensi mosquitoes, collected on day 10, and purified by Accudenz twice, and parasite pellets were stored at -80C. Two million purified oocyst sporozoites were used per parasite line per biological replicate.

RNA preparation RNA from all sample types was isolated by the QIAgen RNeasy Kit (QIAgen, Cat No. 74104) using the manufacturer’s protocol with the additional on-column DNaseI digestion. RNA yields were quantified spectrophotometrically (NanoDrop 2000c, Thermo Scientific), and RNA samples were submitted to the Penn State Genomics Core Facility. The quality of all samples was confirmed by measuring RNA Integrity Number (RIN) using the Agilent Bioanalzyer.

29

RNA-seq A barcoded library was made from each sample by using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, Cat# RS-122-2101) according to the manufacturer's protocol. qPCR was performed to determine the concentration of each library and an equimolar pool was made of all libraries. The library pool was sequenced on an Illumina HiSeq 2500 in Rapid Run mode according to the manufacturer's protocol. For oocyst sporozoite samples, 100nt x 100nt paired-end sequencing was performed, and for gametocyte samples 100 nt single read sequencing was performed.

Transcript identification and abundance determination The resulting data was mapped to the P. yoelii 17XNL strain reference genome (plasmodb.org, v27) using Tophat2 in a local Galaxy instance (version .9) [153]. Gene and transcript expression profiles for both WT and pyalba4- assemblies were generated using Cufflinks (Galaxy version 2.2.1.0), merged into a separate large transcript assemblies with Cuffmerge (Galaxy version 2.2.1.0), and compared using Cuffdiff (Galaxy version 2.2.1.3) [154]. For the purified schizont and gametocyte samples, three biological replicates were merged for

WT and pyalba4- profiles. Oocyst sporozoite samples used two biological replicates for this analysis. Differential transcript abundances were measured in fragments per kilobase of transcript per million mapped fragments (FPKM). All default parameters were used for this analysis, except a masked file containing known non-coding RNAs (available upon request) was applied and the number of maximum fragments per locus allowed was increased from 50,000 to 1,000,000. The final output of this analysis was the differential expression profiles between WT and ALBA4KO for the separate stages analyzed. Statistical analyses carried out by CuffDiff include a Student’s T-test generating a p-value, as well as a multiple test correction generating a q-value. Statistical cutoffs for datasets from each life cycle stage were determined based on overall coverage. For schizonts, entries with a q-value < 0.05 were considered significant. For gametocytes and sporozoites, there were no significant q-values for transcripts other than those encoding YIR proteins. This is likely due to the low percentage of reads that map to the

30

Plasmodium yoelii genome due to contamination of host or vector material. Thus, transcripts with a log2 fold change > 1 and < -1 were also investigated as transcripts-of-interest. These datasets were also compared to the DOZI/CITH RIP-ChIP experiment datasets to conduct targeted analyses [113]. Overlapping transcripts with a significant p-value (<0.01) were considered as hits-of-interest. RNA-seq data reported here is available through the GEO depository (Accession #GSE81834).

31

CHAPTER 3 THE ROLE OF TRANSLATIONAL REPRESSION IN SEXUAL AND TRANSMITTED FORMS OF RODENT MALARIA PARASITES Background It is still unclear whether Plasmodium employs canonical stress granules or a novel RNA storage granule to translationally repress certain transcripts. To further characterize these storage granules and determine if they are or are at least similar to canonical stress granules, immunoprecipitations from Plasmodium gametocytes were performed using GFP-tagged variants of DOZI and CITH [116]. This led to the identification of an important family of DNA/RNA-binding proteins, termed ALBA (acetylation lowers binding affinity) proteins, which have been bioinformatically and experimentally identified in Plasmodium species [114, 118, 130, 131]. Specifically, four ALBA-domain containing proteins have been identified, named ALBA1, ALBA2, ALBA3, and ALBA4, with bioinformatics analyses indicating there may be two more (ALBA5 and ALBA6) [132]. ALBA proteins are derived from an ancient archaeal lineage, and proteins containing ALBA domains are found throughout eukaryotes, including yeast and humans. In yeast, the ALBA domain-containing protein Rpp20 is a subunit of nuclear RNase MRP and P, suggesting a significant role in mRNA homeostasis. In humans, Ribonuclease P protein subunit p25 contains an ALBA domain that aids in tRNA maturation by removing 5'- extranucleotides from the precursor [133]. Interestingly, ALBA4 is apicomplexan specific [132]. When comparing P. yoelii ALBA4’s amino acid sequence to other known protein sequences, it aligns well with notable apicomplexans such as Theileria (50% identity/65% similarity), Babesia (45% identity/60% similarity), Neospora (27% identity/45%similarity), and Toxoplasma 26% identity/42% similarity). PyALBA4 also aligns with a chromerid, Vitrella (42% identity/61% similarity), which is thought to be an evolutionary ancestor of the apicomplexan lineage [134]. This may indicate PyALBA4’s functions are also evolutionarily ancient and conserved within this specific lineage. The hypothesized ancient function of ALBA proteins is broadly to bind nucleic acids. In Sofolobus, ALBAs are shown to coat DNA and possibly contribute to chromatin organization, whereas in the eukaryotic lineage they are predicted to play a role exclusively in RNA

32 metabolism [87]. The structure of the ALBA domain shares a high structural similarity with the E. coli Yhby protein, a characterized member of the chloroplast RNA splicing and ribosome maturation (CRM) RNA-binding protein family [135]. In E. coli, Yhby is associated with pre-50S ribosomal units, indicating a role in ribosome assembly that could affect translation [135]. In terms of protein-protein interactions, ALBA proteins are known to homo- and heterodimerize with each other [87, 132, 136, 137], which may allow for redundant or enhanced function. These possible functions of ALBA proteins have been studied in other protozoan parasites, including Leishmania and Trypanosomes. In these species, ALBA proteins localize to cytosolic RNA granules that interact with developmentally important transcripts [138-140]. In Trypanosomes, ALBA proteins are particularly important for proper progression from stage- to-stage in the parasite life cycle. As the parasite transitions from the mesocyclic to the epimastigote stage (Figure 3), ALBA3 and ALBA4 expression is depleted, suggesting ALBA proteins aid in developmental

Figure 3. Trypanosome life cycle transitions. When ALBA3 and ALBA4 are knocked out in the procyclic phase, the parasites morphological resemble those undergoes the mesocyclic to epimastigote transition. Excitingly, ALBA proteins localize to cytoplasmic RNA granules in response to nutrient deprivation [139], reminiscent of a canonical stress granule under a classic stressor. In the absence of ALBA3 and ALBA4, two interesting phenotypes are observed. The first is ‘termed’ nozzle, wherein the organism develops an elongated posterior end, in some cases reaching up to 50% increase in length [139]. A similar phenotype is observed when TbZFP2, a zinc finger protein hypothesized to bind RNA, is overexpressed. This suggests TbZFP2 may play a critical role in determining when the parasite enters into various stages of development, as this overexpression leads to cells accumulating in G1 phase. Hypotheses

33 explaining this arrest include perturbations in cell-cycle machinery or the cytoskeletal prevent cell-cycle progression, ultimately stunting the parasite [141]. The second phenotype Subota et. al. observed was the nucleus in a posterior position relative to the kinetoplast. These two phenotypes are consistent with ALBA-domain containing proteins’ involvement in developmental transitions. In Plasmodium species ALBA proteins from P. falciparum bind to both DNA and RNA, and also localize to cytoplasmic RNA granules that are nuclear adjacent during the ring stage, but become distributed throughout the cytoplasm in later blood stage parasites [114, 118, 131]. Deeper characterization of PfALBA1 recently elucidated its role in translational repression and RNA regulation during the asexual trophozoite blood stage [114]. PfALBA1 contains the ALBA domain and a RGG domain, another known RNA-binding domain, and classic marker of some stress granule proteins. Vembar and colleagues found PfALBA1’s association with certain mRNAs leads to their translational repression, while PfALBA1’s dissociation from these transcripts leads to their translation [114]. In rodent infectious species, ALBA proteins associate with the DOZI/CITH complexes, indicating they may also play a role in translational repression in Plasmodium [116]. These observations suggest protozoan parasites have evolved RNA-binding proteins and RNA granules to play critical roles in RNA homeostasis and posttranscriptional regulation prior to transmission between vector/host and host/vector, and during life cycle transition events. Though still contentious, there is mounting evidence ALBA proteins are a member of canonical stress granules in protozoan parasites. However, current understanding of the essentiality and roles of ALBA proteins in transmission, especially the apicomplexan-specific ALBA4 protein, is limited. To this end, the first Plasmodium ALBA knockout line was generated by genetically disrupting alba4 in P. yoelii (pyalba4-) to determine the importance of PyALBA4 in both gametocytes and sporozoites. Morphological and behavioral phenotypes were observed in each of these life cycle stages – points at which translational repression and mRNA stability are critical [113, 115]. Additionally, comparative total RNA-seq with wild-type parasites indicates RNA homeostasis for specific transcripts is perturbed in knockout parasites. Using a C- terminally tagged variant of PyALBA4 to visualize the spatial arrangement of PyALBA4 revealed

34

PyALBA4 is found in cytosolic, nuclear-adjacent, and plasma membrane-adjacent granules. The varied localizations of the protein are consistent with a role in affecting RNA metabolism in multiple ways depending on its spatial distribution and interacting proteins. Therefore, it is hypothesized PyALBA4 has stage-specific functions; in gametocytes and sporozoites, PyALBA4 is associated with a canonical stress granule. In asexual stages, PyALBA4 is a generalist aiding in multiple facets of mRNA homeostasis including mRNA nuclear-to-cytoplasmic transport, active translation, mRNA degradation, and canonical stress granules. Until recently, biochemical analyses in sporozoites have proven difficult; interrogation of a single protein has only been possible through autoradiography and western blotting approaches. Attempts to robustly identify multi-protein complexes have been lacking. This technical intractability is due in part to the massive contamination of sporozoites with proteins, nucleic acids, and lipids from the mosquito vector following microdissection. This problem is well-known in the field, and efforts to conduct large-scale transcriptomic and proteomic analyses of sporozoites have been greatly restricted due to these mosquito contaminants [103, 142]. Described herein is a minimally perturbing, scalable method to purify large numbers of sporozoites, which has enabled comprehensive transcriptomics and proteomics and surface proteomics [106, 143, 144]. For the first time, this novel sporozoite purification approach has been utilized to purify large numbers of oocyst sporozoites, allowing interrogation by both comparative RNA-seq and by a biotin/streptavidin-based immunoprecipitation method to identify a cytosolic complex bound to PyALBA4. Moreover, this approach has been adapted to also identify proteins that associate with PyALBA4 in multiple life cycle stages, including gametocytes, asexual schizonts, and sporozoites. This experimentation revealed PyALBA4 consistently associates with specific proteins regardless of stage, but also associates with distinct, stage-specific effector proteins. In gametocytes, a translationally repressive complex is detected. In schizonts PyALBA4 interacts with multiple complexes, including repressive and active translational machinery, mRNA export, and NPGs and/or P-bodies. Excitingly, in sporozoites, PyALBA4 interacts with other ALBA proteins, potentially forming a complex involved in translational repression at this critical transmission stage. This work demonstrates the importance of RNA-binding proteins and RNA homeostasis

35 in proper parasite development and transmission. While some of these findings are consistent with canonical stress granules, without further experimentation it is not possible to determine if all the complexes identified herein are canonical stress granules. This is further complicated by the dynamic nature of mRNPs, stress granules, and P-bodies. However, the leading hypothesis is this is a likely mechanism employed by the parasite. The existence of stress granules, canonical or otherwise, is exciting, as it opens these parasites to further intervention and/or exploitation.

Results PyALBA4 knockout parasites exhibit defects in gametocytes and sporozoites Because RNA-binding proteins (RBPs) such as Puf2 and DOZI/CITH play important roles in the transmitted forms of the parasite (gametocytes and sporozoites), the importance of a family of RBPs that is functionally associated with them, ALBA proteins, was explored [116]. To do so, a knockout parasite line was generated by homologous double crossover recombination for the apicomplexan-specific P. yoelii alba4 gene, wherein GFPmut2 and HsDHFR cassettes replaced the entire open reading frame of pyalba4 (pyalba4-). The integration event was confirmed by Figure 4. pyalba4- clonal populations were GFP fluorescence (data not shown) and genotyping generated by double homologous crossover PCR (Figure 4). Two independent clonal populations recombination and confirmed by genotyping PCR. A) The entire pyalba4 ORF was deleted were also generated, through limited dilution cloning. by introducing linear plasmid DNA containing two homology repair sequences flanking a GFP When compared to a wild type parasite line cassette and HsDHFR cassette. B) The successful disruption of the pyalba4 ORF was expressing GFPmut2 from a dispensable locus (WT- confirmed by genotyping PCR, and two clonal parasite lines were isolated by limited dilution GFP), pyalba4- parasites exhibited a defect in sexual cloning. stages, namely in male gametocytes. These lines showed a two-fold increase in the number of exflagellating males per microscopic field,

36

Figure 5. pyalba4- parasites are adversely affected in gametocyte and sporozoite stages. The entire life cycle was characterized and compared to a WT- GFP line known to behave as WT. A) Exflagellating male gametes were identified by centers of movement (COM) and quantified per 40X field of vision. pyalba4- parasites exhibit a two-fold increase in the number of COMs compared to a WT- GFP control. To quantify this, 10 fields of vision per parasite line per biological replicate were analyzed, and six biological replicates were used. B) The fraction of total sporozoites found in the salivary glands was determined by performing double dissections of the midguts and salivary glands of each mosquito. In WT-GFP parasites, a majority of the sporozoites are found in the salivary glands by Day 14 post blood meal. In contrast, most pyalba4- sporozoites remain associated with the midgut of the mosquito throughout the infection of the mosquito. Three biological replicates per parasite line were assessed, with 10-25 mosquitoes per parasite line per time point. Student’s t-test, *pvalue < 0.05, **p-value < 0.01. indicating PyALBA4 may play a role in gametocytogenesis and/or male gamete activation (Figure 5A). To rule out global changes in gametocytemia or the male-to- female sex ratio, gametocytemia was assessed by thin blood smears fixed with methanol and treated with Giemsa stain. Gametocytes were assigned into three major categories: sexually dimorphic gametocytes (immature), mature males, and mature females, as has been previously reported [138]. To facilitate scoring of P. yoelii gametocytes, a visual key of Giemsa-stained P. yoelii parasites was constructed based upon previous observations with P. berghei (Figure 6) [138]. No differences in overall gametocytemia, sex ratio, or percentage of immature gametocytes were observed, leading to the conclusion that PyALBA4 does not play a role in global gametocyte commitment or sex determination (Table 1). Rather, these data indicate PyALBA4 plays an important role in the activation of existing male gametocytes. Interestingly, in the absence of PbPuf2 there is also an increase in exflagellating males [98]. These observations indicate RNA-binding proteins are important in both male and female gametocytes, and provide evidence that PyALBA4 plays an important role in the regulation of male gamete activation. In the other transmitted form of the parasite, sporozoites, the abundance of transcripts encoding for PyALBA proteins are affected by the genetic deletion of PyPuf2 [106]. This leads to a hypothesis wherein PyALBA proteins are also important to sporozoite development and

37 transmission. Therefore, pyalba4- parasite lines were characterized throughout the life cycle, including mosquito and liver stages of development. No difference in the prevalence of infection of mosquitoes, defined as percentage of mosquitoes infected (Table 2), nor any statistically significant difference in the number of oocysts on Days 7 and 10 post blood meal as compared to WT-GFP parasites (Figure 7) were observed. There is also no statistically significant difference in the total number of sporozoites found per infected mosquito, which was determined by dissections of both the midguts and salivary glands of the same mosquito (Figure 8). These data suggest the absence of PyALBA4 does not adversely affect transmission to mosquitoes or the establishment of the mosquito infection. However, on Day 14 post blood Figure 6. Scoring key for Plasmodium yoelii meal, when WT-GFP sporozoites have semi- gametocytes. Plasmodium yoelii parasites were binned into synchronously egressed from midgut oocysts and three categories: immature gametocytes, mature females, and mature males. Mature females were invaded the mosquito salivary glands, a majority of identified by condensed nuclear staining, and - development within the entire RBC. Additionally, the pyalba4 sporozoites remain largely associated osmiophilic bodies are also present in mature female gametocytes. Mature male parasites were with the mosquito midgut (Figure 5B). Over time, characterized by a slightly pinker stain, less - condensed nuclear staining, and an amorphous pyalba4 sporozoites do proceed to invade the shape. Immature gametocytes were characterized by intermediate nuclear staining and did not salivary gland but appear to do so asynchronously occupy most or all of the RBC. and at a greatly reduced rate. Interestingly, the sporozoites that do reach the salivary glands are fully infectious, as the time to achieve blood stage patency (defined as two or more infected RBCs per 20,000 RBCs) was identical to WT-GFP (Table 3). These data suggest PyALBA4 is also important for the semi-synchronous development of sporozoites and their movement from the oocyst to the salivary gland. This lack of synchronicity is potentially due to perturbation of the preferred RNA homeostasis, which

38 perhaps can be sufficiently overcome by the relatively small fraction of sporozoites that can egress from the midgut and invade the salivary gland.

Figure 7. pyalba4- parasites do not exhibit significant changes in the number of oocysts per infected mosquito. A) Oocyst numbers per infected mosquito were counted on Day 7 post blood meal in both the WT- GFP line and pyalba4- lines. Mosquito midguts from 20 mosquitoes were dissected and placed on a slide and gently covered with a coverslip. Oocysts were counted by fluorescence microscopy. There is no significant change in the number of oocysts per infected mosquito. B) Oocysts were also quantified Day 10 post blood meal for each parasite line. Using 20 mosquitoes per group, there was no significant change in the number of oocysts per infected mosquito. Three biological replicates are shown, presented as the average with the associated standard error.

39

Figure 8. The total number of sporozoites per infected mosquito remains unchanged in pyalba4- parasites. The total number of sporozoites per mosquito was calculated for each parasite line. There was no significant change in the total number of sporozoites found per mosquito. Three biological replicates are shown, with 10-25 mosquitoes per group per replicate, and presented as the average with the associated standard error.

PyALBA4 expression in transmitted forms is consistent with a RNA-granule In order to further investigate PyALBA4’s function, a transgenic parasite line expressing a full-length variant of PyALBA4 with a GFPmut2 tag appended to its C-terminus (PyALBA4::GFP) was used. The genetic modification was confirmed by genotyping PCR (Figure 9), live fluorescence, and indirect immunofluorescence assay (IFA) of GFP Figure 9. PyALBA4::GFP parasites were generated by expression. No morphological or behavioral double homologous crossover recombination and confirmed by genotyping PCR. defects with this line compared to WT-GFP were A) A PyALBA4::GFP transgenic line was generated by introducing linear plasmid DNA containing two observed. In sexual blood stages, PyALBA4::GFP homology repair sequences directed to the C-terminal end of the protein and its 3’ UTR flanking a GFP-tag and is expressed diffusely throughout the cytoplasm HsDHFR cassette. Two populations of transgenic parasites were generated (Pop. 1 and Pop. 2). B) The of male gametocytes with some regions of successful addition of the GFP-tag was confirmed by genotyping PCR.

40 slightly higher concentration. In female gametocytes, PyALBA4::GFP expression is largely diffuse with few punctate foci (Figure 10B). This was surprising, as ALBA proteins associate with DOZI, which exhibits a more strongly punctate staining pattern seen in P. berghei female gametocytes [107]. This localization suggests PyALBA4 may be participating in other complexes and processes beyond its appreciated role in the DOZI/CITH/ALBA complex. In the mosquito stage of development, PyALBA4::GFP expression and localization was assessed by live fluorescence and IFA. PyALBA4 is expressed throughout mosquito stage development, although its localization changes over time. In early oocysts (Day 3 post-blood meal), PyALBA4::GFP expression is exclusively diffuse throughout the entire oocyst (Figure 11). As oocysts mature and sporozoites form, two expression patterns emerge: punctate expression and diffuse expression. In oocyst sporozoites liberated from the midgut (Day 10 post-blood meal), both dominant, cytoplasmic foci and some diffuse expression throughout the cytoplasm persist (Figure 10C). Finally, expression of PyALBA4::GFP in salivary gland sporozoites (Day 14 post-blood meal) is very similar to oocyst sporozoites, with a stronger localization to punctate foci, though some diffuse expression is still present (Figure 10C). Because the formation of cytoplasmic foci is present, it is possible PyALBA4::GFP is localizing to a RNA-containing protein complex (mRNP). Low-level diffuse expression may indicate PyALBA4::GFP is available in the cytoplasm for processes and complexes other than those with mRNA-storage complex functions, as observed in female gametocytes. Taken together, the timing of these PyALBA4::GFP-containing cytoplasmic foci that form during sporozoite development in the oocyst and salivary gland matches the timing of the phenotype attributed to the genetic disruption of PyALBA4, and thus may be functionally linked.

41

Figure 10. PyALBA4 is expressed throughout the life cycle and is found in multiple subcellular locations. PyALBA4::GFP localizes to punctate foci and/or diffusely through the cytoplasm. A) In asexual blood stage, PyALBA4::GFP expression is exclusively nuclear-adjacent in rings, but includes cytoplasmic punctate foci in schizonts, with an intermediate expression pattern in trophozoites. In trophozoites, localization also appears along the plasma membrane. Asexual stage IFAs were performed with anti-GFP and counterstained with anti-ACP. Nuclei were stained with DAPI. B) In male and female gametocytes expression is cytoplasmic and diffuse, with areas of greater intensity. In female gametocytes these areas of greater intensity overlap with DDX6 (DOZI) expression. Female gametocyte IFAS were performed with anti-GFP and counterstained with anti-DDX6, while males were counter-stained with anti-alpha tubulin II. Nuclei were stained with DAPI. C) In mosquito stages, both oocyst- and salivary gland sporozoites exhibit punctate cytoplasmic expression, as confirmed by both immunofluorescence assay and live fluorescence. There is also some slight diffuse cytoplasmic expression seen in both stages. Both stages were stained with anti-GFP, counterstained with anti-CSP, and nuclei were stained with DAPI for IFAs.

Figure 11. PyALBA4::GFP is expressed throughout mosquito stage development, and exhibits two distinct expression patterns in oocysts. A) Midguts were dissected from infected mosquitoes at several time points post blood meal and oocysts were assessed by fluorescence microscopy. Early oocysts (Day 3) exhibit a single PyALBA4::GFP diffuse expression pattern. Throughout oocyst development (Day 5-Day 10), two distinct expression patterns emerge: diffuse, and punctate foci. These occur in a roughly 1:1 ratio, and punctate foci appear to segregate into budding sporozoites. 42

PyALBA4 associates with many canonical stress granule components in gametocytes DOZI, CITH and Puf2 RBPs play critical roles in translational repression and localize to cytoplasmic punctate foci in both gametocytes and sporozoites [98, 106, 107, 115, 116, 118, 127, 131, 155]. DOZI/CITH complexes also interact with ALBA proteins, which are likely required to provide additional functionalities to translationally repress the recruited mRNAs. However, because the genetic deletion of DOZI causes a complete arrest in development at the zygote stage, no exploration of the function of DOZI/CITH/ALBA-containing storage granules beyond this point in the life cycle has been reported. Therefore, it is unknown if translational repression by this particular complex occurs, or if Puf2-containing granules or other discrete granules provide all necessary translational repressive functions. Because PyALBA4 plays important roles in both gametocytes and sporozoites as evidenced by the observed knockout phenotypes, it is hypothesized PyALBA4 associates with a translationally repressive complex in both gametocytes and sporozoites. However, it is intriguing to consider these complexes might have different protein compositions in the various life cycle stages where PyALBA4 is expressed (asexual, gametocyte, sporozoite). To determine if stage-specific and stage- independent compositions of the PyALBA4 complex exist, the GFP tag on the PyALBA4::GFP protein variant was used as a handle to perform immunoprecipitations coupled with nano LC/MS-MS (IP/MS) to identify proteins binding directly or indirectly with PyALBA4. Gametocyte samples from either a WT-GFP or PyALBA4::GFP line were generated by treatment with medicated water containing 10mg/L sulfadiazine, which effectively killed all asexual parasite forms in infected mice. Following purification by a discontinuous Accudenz gradient, gametocyte samples were cross-linked with formaldehyde to preserve any transient or weak protein:protein interactions and to allow for stringent washing conditions. Samples were then lysed chemically, mechanically and by sonication. Complexes containing PyALBA4::GFP were captured by introducing streptavidin-coated beads coated with a biotin- conjugated anti-GFP antibody to parasite lysates. Efficient capture of PyALBA4::GFP was monitored at key points throughout the immunoprecipitation by collecting samples for bookkeeping purposes (e.g. 5% Input, Flow-Through, and final elution after cross-link reversal). A western blot was first performed with each of these samples to confirm successful pull-down

43 of GFP (26kD) and PyALBA4::GFP (69kD) from the WT-GFP and PyALBA4::GFP parasite lines, respectively (Figure 12A). There are also higher molecular weight bands, which may be a result of incomplete reversal of the formaldehyde crosslinks, as well as a light band seen slightly higher than 25kDa in the PyALBA4::GFP elution, which indicates that a possible cleavage event near the C-terminus of PyALBA4 is occurring (Figure 12). These data indicate that both GFP and PyALBA4::GFP were successfully captured in sufficient quantities to allow for downstream proteomic analyses.

Figure 12. PyALBA4::GFP associates with translational repression machinery and active translational machinery in a largely asexual population, as assessed by immunoprecipitation and nano LC/MS/MS. Following immunoprecipitation of PyALBA4::GFP, cross-links were reversed, and samples were used for immunoblotting to confirm capture of PyALBA4::GFP and assess efficiency of the cross-link reversal. A) Immunoblotting with anti-GFP of the 2.5% Input, Flow Through (FT), and Elution samples of the WT-GFP and PyALBA4::GFP gametocytes was done. PyALBA4::GFP is ~69 kDa and GFP is ~26kDa, and both run as expected and are clearly seen in the elutions. GFP is notably absent from the WT-GFP Input, as this is a very dilute sample. High weight molecular bands were seen, indicating incomplete cross-link reversal. A recombinant GFP with a 6XHis-GST tag (~55kDa) was used as a positive control. B) Immunoblotting was performed as previously mentioned with WT-GFP and PyALBA4::GFP schizont samples. GFP is again absent. High weight molecular bands were seen, indicating incomplete cross-link reversal. A recombinant GFP with a 6XHis-GST tag (~55kDa) was used as a positive control. To robustly determine the composition of PyALBA4 complexes, three biological replicates were performed and confirmed by immunoblotting as described above. The remaining elution samples were then processed and subjected to nano LC/MS-MS (IP/MS) to identify PyALBA4-binding proteins. Immunoprecipitations from the WT-GFP line were done in parallel to control for proteins that bind to the beads, antibody, and/or GFP. Bioinformatic analysis tools contained in the Trans-Proteomic Pipeline were used to identify peptides and infer protein identities, and a list of proteins below a false discovery rate (FDR) of 1% are reported (Table 4,5*) [146]. To confidently determine the validity of protein identifications above this threshold and across replicates, replicate datasets were analyzed by SAINT, which compares normalized spectral count and protein length data [152]. Following this analysis, protein identifications

44 below a 10% FDR were considered robust hits in these datasets, as previously described [144]. These stringent thresholds will likely result in the underreporting of some true positives present in lower abundances (Table 5*, available in online repository; protein identifications with >10% FDR are bolded) despite their known roles in translational repression (e.g. Puf1, DCP2, HSPs, eIFs). Confident inclusion of these would require further validation.

As ALBA proteins are known to interact with other ALBA proteins and the DOZI/CITH complexes, members of the DOZI/CITH complexes were expected to be identified [116]. There were 33 proteins in gametocytes with stringent statistical cutoffs identified that interact with PyALBA4 (Table 4). Of these proteins, 23 of the orthologous proteins in P. berghei are also known to interact with the DOZI and/or CITH complexes (Table 7*, Figure 13A) [116]. These components include proteins canonically involved in stress or storage granules, including DOZI

45 and CITH themselves, other ALBA proteins (PyALBA1, PyALBA2, PyALBA3), poly-A binding protein (PABP), eukaryotic initiation factors such as eIF4E and eIF4A, 40S ribosomal proteins (PY17X_1314600, PY17X_1334000), other known RNA-binding proteins (HoBo and HoMu). Several uncharacterized conserved proteins were also identified (PY17X_0705000, PY17X_1116400, PY17X_0521000, PY17X_1117900). Another protein-of-interest found is PY17X_1208200, a predicted RNA-binding protein likely to be Guanylate Binding Protein 2 (GBP2) based on previous bioinformatic analyses [132]. GBP2 is also a known component of stress granules which plays a role in shuttling between the nucleus and cytoplasm [159]. However, many of the proteins identified in the DOZI and CITH immunoprecipitation experiments are also observed here. These observations suggest that PyALBA4 is involved in a translationally repressive complex, but likely serves another function in mRNA processes.

Figure 13. PyALBA4::GFP associates with multiple complexes in a stage specific manner, with differing associations in gametocytes and schizonts. PyALBA4::GFP schizonts or gametocytes were collected by discontinuous gradient as mentioned previously, and chemically cross-linked with formaldehyde. Lysates from either GFP-only or ALBA4::GFP parasites were pre-cleared on empty beads followed by an incubation with streptavidin beads coated with a biotin-conjugated GFP antibody. Proteins were bioinformatically analyzed through the TransProteomic Pipeline (TPP), and replicates were combined by SAINT. A) A comparison of the proteins associating with PyALBA4 in gametocytes and those associating with the DOZI/CITH complexes was completed. Thirty-three proteins were pulled down with PyALBA4::GFP, with significant overlap with the DOZI/CITH complexes. B) A comparison of the proteins that associate with PyALBA4 in gametocytes and schizonts was performed. Most proteins identified to interact with PyALBA4 do not overlap between stages. C) A comparison of the proteins that associate with PyALBA4 in schizonts and those that associate with the DOZI/CITH complexes was completed. One hundred and thirty three were pulled down with PyALBA4::GFP, with some overlap with the DOZI/CITH complexes. However, 88 protein interactions are specific to PyALBA4 in schizonts. These findings suggest the role of PyALBA4 in gametocytes is multi-faceted and dependent on binding partners. When PyALBA4 is associated with DOZI/CITH in a complex (referred to as the DOZI/CITH/ALBA (DCA) complex), this cytosolic complex may be acting as a storage granule to repress and preserve specific mRNAs [107, 113, 115, 116]. This model is

46 supported by the similarity of the composition of DCA complexes with canonical stress granules [47]. For instance, several eukaryotic elongation factors (EF1alpha and beta, EF2) and initiation factors that participate in yeast and mammalian stress granules are detected, including eIF4A and eIF4E [204]. In addition, eIF4G was not detected. eIF4G is an important scaffolding protein that effectively circularizes mRNAs by binding to both the cap-binding protein (eIF4E) and poly- A binding protein (PABP1). The absence of eIF4G may be explained in part by the presence of Musashi, which is a RBP known to inhibit translation by directly binding polyuridine-rich sequences in 3’ UTRs of mRNAs and subsequently blocking the PABP1 and eIF4G interaction [160]. Excluding eIF4G from mRNAs to prevent circularization to reduce translational efficiency is a common mechanism in other species, and Plasmodium may also use this to repress translation [161]. The abundance of PABP1 in gametocyte samples supports the idea that the DCA complex functions as a storage granule complex, as it is one of the canonical members of stress granules in yeast. Interestingly, Plasmodium encodes two poly-A binding proteins: PABP1, which is cytosolic, and PABP2, which is observed in both nuclear and nuclear-adjacent compartments (unpublished results). In gametocytes, only PABP1 was identified as an interacting partner with ALBA4. PABP2 associates with NPGs, thus its absence here is consistent with DCA acting as a stress or storage granule in gametocytes. Lastly and importantly, no large ribosomal subunits were detected. As these are loaded to initiate translation and upon binding to transcripts, they may inhibit the trafficking of these mRNAs into stress granules [162]. Translational repressors that prevent loading of 60S ribosomes, such as the well-characterized ZBP1 protein, can translationally silence the bound transcript [163]. Though this protein is not detected, it is possible another uncharacterized protein serves this function. The small ribosomal subunits detected are consistent with their role in stress granules in Plasmodium and in model eukaryotes [43, 116]. Their presence also supports the model that these granules, upon receiving the proper stimuli, can immediately resume translation, which is consistent with the prevailing models of studies in eukaryotes. Similar to the DOZI/CITH complexes, PyALBA4 also associates with splicing factors, housekeeping proteins, and uncharacterized proteins. These include the CUG-BP- and ETR-3-

47 like factor (CELF) family proteins, which are involved in RNA-binding and splicing. In gametocytes, both CELF2 (also referred to as Bruno or HoBo) and CELF4 (PY17X_1453200) were detected. CELF family proteins traffic between the nucleus and the cytosol, which may explain their association with the complex [175]. Several different housekeeping proteins were also detected across DOZI/CITH and ALBA complexes, though it remains unclear if these are bona fide interactions or driven by their abundance. It is also possible housekeeping proteins play some yet-to-be-defined role in RNA metabolism. For instance, ALDO2, GAPDH, enolase (ENO), and heat shock proteins are detected here as well as in DOZI and CITH complexes and hypothesized to aid in stress resolution. Additionally, four uncharacterized proteins are shared across DOZI/CITH and ALBA4 complexes (Table 7*). As there are no predicted domains or putative functions for these proteins, characterization of these proteins may further elucidate and refine the function of these complexes. Taken together, the composition of these complexes strongly suggests a clear role for PyALBA4 in a stress granule in gametocytes, which is necessary for the parasite to protect and translationally repress specific transcripts required for establishment of infection following transmission. However, lower abundance proteins were also detected, suggesting PyALBA4 is also interacting with newly exported mRNAs, perhaps prior to their fate determination. Newly matured mRNAs are exported from the nucleus to the cytoplasm and are often still bound by splicing factors [164]. These splicing factors and their associations with mRNAs can direct their fate. One such fate is localization to a stress granule [43, 165], as described above. However, several splicing factors associate with PyALBA4 that have not been previously detected in DOZI/CITH complexes. This is consistent with PyALBA4’s association with a nascent cytoplasmic complex. These include pre-mRNA-splicing factor SR1 (Py17X_1235500), RAN/TC4 (PY17X_0932300), and PRE-binding protein (PREBP, PY17X_1213400). SR1 is a pre-mRNA splicing factor shuttles between the nucleus and the cytosol. Interestingly, PfSR1 is nuclear- adjacent throughout blood stage, but not nuclear [166, 167]. Ran/TC4, a GTP-binding nuclear protein (PY17X_0932300), is involved in transport of RNA and proteins into and out of the nucleus via the nuclear pore complex. PRE-binding protein (PREBP) (PY17X_1213400) is possibly a transcription factor in Plasmodium, however, the S3 family of ribosomal proteins contain the

48 same domain found in PREBP [168]. The S3 family of ribosomal proteins includes ribosomal proteins that are implicated in the structural rearrangement of small ribosomal units, which allows mRNAs to enter and scanning to occur [169]. This suggests a possible role in translation. Taken together, associations with these proteins and the diffuse localization of PyALBA4 in gametocytes indicates PyALBA4 may bind to mRNA complexes as they reach the cytoplasmic interface from the nucleus and remain associated with these mRNAs as they traffic to storage granules or other areas of the cell. It is important to note there are other non-overlapping members of the PyALBA4, DOZI, and CITH complexes (Figure 13A Table 7). These include cytoskeletal proteins known to function in active transport of granules throughout the cell, which may influence activation of gametocytes as evidenced by the phenotype in activated male gametes [170]. Interestingly, two conserved unknown proteins with no predicted domains (PY17X_0806800, PY17X_1117900) specific to the PyALBA4 complex were detected. Further characterization of these proteins may illuminate other functions specific to PyALBA4 when it is not in a DCA complex. Taken together, in gametocytes PyALBA4 is associating with proteins related to RNA processing and translational repression as well as translational proteins known to remain associated with mRNA in repressive granules.

PyALBA4 interacts with proteins related to its gametocyte phenotype Interestingly, the presence of calcium-dependent protein kinase 4 (CDPK4) in the ALBA4 complex may partially explain why genetic disruption of PyALBA4 led to an increase in the activation of male gametocytes. CDPK4 is a Ca2+-based regulator of signaling and is critical for male gamete activation, as exflagellation is blocked in the presence of bumped kinase inhibitors (BKIs) [171]. CDPK4 localization is largely limited to the periphery of the cell [138, 172], which is consistent with PyALBA4 location during the trophozoite stage (Figure 10A). In schizonts and gametocytes, CDPK4 is cytoplasmic but more diffuse. Additionally, although a gross phenotype in female gametocytes is not seen when PyALBA4 is knocked out, an interaction between PyALBA4 and NIMA-related kinase 4 (NEK4), which is essential in female gametocytes for proper development of parasites in the mosquito host [173, 174], is detected. Therefore, it

49 is still possible female pyalba4- gametocytes are affected but perhaps in a subtler manner. It is intriguing to consider PyALBA4 is associated with these two proteins important in gametocytes, and an attractive hypothesis is PyALBA4 acts to sequester these as a regulatory mechanism of gametocyte activation.

PyALBA4 associated proteins are post-translationally modified in gametocytes ALBA proteins were originally defined by the effects post-translational modifications (PTMs) have upon their ability to bind to nucleic acids (Acetylation Lowers Binding Affinity). Although samples were not subjected to enrichment for any PTMs, it was possible to confidently identify modified proteins in both gametocyte and schizont samples. A simple comparison between detected proteins and a database consisting of only acetylated or phosphorylated protein sequences was performed. No acetylation modifications were detected in unenriched peptide samples, though it is unclear if acetylated proteins are not present or simply were not detected due to lack of enrichment. However, phosphorylation is readily detected on peptides from gametocyte samples (Table 8). Notable phosphorylation events included Bruno/CELF2 (residue S83 or S84), PyALBA3 (S3 or T4), and GBP2 (T9). Post-translational modifications, such as phosphorylation, can be important for binding to and determining the fate of mRNAs. This is the case with some splicing proteins, which rely on (de)phosphorylation for association/release from newly exported mRNAs [165]. As discussed earlier, it may also prove to be critical for canonical stress granule assembly/disassembly. This further supports the hypothesis that in gametocytes PyALBA4 is part of a canonical stress granule.

50

PyALBA4 affects translationally repressed transcripts in gametocytes The absence of PyALBA4 led to a clear gross phenotype in male gametes, and IP/MS data indicates PyALBA4 is likely acting in a translationally repressive stress granule in gametocytes (Table 4). In agreement with this, most transcript changes observed in pyalba4- parasites were decreases in transcript abundance, congruent with PyALBA4 playing a protective and translationally repressive role in gametocytes. There were 535 transcript abundances observed with a greater than 2-fold change when compared to WT-GFP parasites. The majority (309) of these transcripts encode for currently uncharacterized proteins. Unfortunately, though they may be vital components of this regulatory network, they are excluded from this analysis due to lack of information on their translation and resultant proteins. Of the remaining 226 transcript abundance changes, only 36 transcript abundance increases in pyalba4- parasites were observed when compared to WTGFP controls, while 190 transcript abundance decreases were seen (Table 9*). The most notable transcripts with an increase in transcript abundance include a calcium-binding protein (PY17X_0105700) and a calcyclin binding protein (Py17X_0837400). While these proteins have

51 not been directly linked to the activation of gametes, calcium-regulating proteins such as CDPK4 are known to be important, and thus these may contribute to the observed exflagellation phenotype [171, 172].

Transcript abundance decreases observed in pyalba4- gametocytes include transcripts of proteins involved in the stages of the life cycle that follow (Table 7*). LCCL domain-containing proteins (CCp1, CCp2, CCp4, LAP5), which are released during gametogenesis and actively surround newly emerged micro- and macro-gametes, were observed [179]. Interestingly, disruption of pfccp2 does not affect the parasite’s development through oocyst sporozoite formation, yet salivary gland sporozoites are not present in infected mosquitoes [179]. The transcript abundance decrease of pyccp2 may contribute to the asynchronous release of oocyst sporozoites phenotype in pyalba4- sporozoites. The transcripts of three members of the CPW- WPC protein family are also differentially expressed, notably, as they are known to be translationally repressed until the zygote and ookinete stages [180]. These data further support the hypothesis that PyALBA4’s primary function in gametocytes is to contribute to translational repression of specific mRNAs. Notable transcript decreases include those which encode for proteins important for ookinetes, such as von Willebrand factor A domain-related protein (WARP), PSOP6, 13, and 20, and several transcripts associated with gliding motility (GAPM2, GAP45, IMC1b-IMC1k, and GAP50). As gliding motility is a critical function of the ookinete and directly affects its ability to productively infect the vector, these decreases are relevant. While no gross defects were observed until the oocyst sporozoite stage, it is possible the parasite is able to overcome any small deficits in these transcripts, and/or that less pronounced phenotypes were not observed. To further understand if PyALBA4 is acting in a canonical stress granule in gametocytes, pyalba4- transcript abundance changes were compared to the DOZI/CITH RIP-ChiP dataset, wherein specific transcript targets of the DOZI/CITH complexes were determined (Figure 14C, Table 10*). Unsurprisingly, 108 transcripts detected in pyalba4- parasites overlap between the datasets. These include transcripts important in female gametocytes as well as the following ookinete stage. A transcript of interest encodes for an ApiAP2 transcription factor (PY17X_1317000), which interacts with PbDOZI and PbCITH [113], and shows a decrease in

52 transcript abundance in pyalba4- parasites (-2.4 fold change). This is consistent with the assertion PyALBA4 is playing a role in translational repression in gametocytes. However, a majority of the transcript abundance changes in pyalba4- parasites are specific to this dataset. A GO term analysis was performed on these specific transcripts, but no confident GO terms were called. Manual inspection of the list revealed many of these transcripts encode conserved yet unknown protein products. Therefore, PyALBA4 contributes to translational repression of specific mRNAs found within DCA complexes in gametocytes, and characterization of the roles these regulated mRNAs may contribute to parasite transmission is warranted.

Figure 14. Comparison of pyalba4- transcript abundance changes with related published datasets. The pyalba4- comparative total RNA-seq datasets from schizont and gametocytes were compared to transcripts that interact with PfALBA1 (A), transcripts known to be translationally repressed in asexual stage (B), and DOZI/CITH targeted transcripts (C). A) Transcripts that are regulated by PfALBA1 in late trophozoite stages were compared to pyalba4- schizont transcript abundance changes. There is very little overlap between these groups though similar categories of mRNA transcripts are affected, including transcripts important in invasion. B) Known translationally repressed transcripts in asexual Plasmodium stages were compared to pyalba4- schizont transcript abundance changes. Again, there is little overlap, indicating PyALBA4’s major function in schizont and asexual blood stage may not be participation in translational repression. C) Transcripts known to interact with PbDOZI and PbCITH in female gametocytes were compared to pyalba4- gametocyte transcript abundance changes. There is notable overlap of female gametocyte specific and ookinete-related transcripts, which is consistent with the known DOZI/CITH/ALBA4 interaction. Transcripts specific to the pyalba4- dataset encode for mostly conserved unknown proteins.

PyALBA4 associates with PyALBA1 and PyALBA2 in oocyst sporozoites As Plasmodium undergoes two transmission events in its life cycle (host-to-vector as a gametocyte, vector-to-host as a sporozoite) and expresses the same proteins involved in translational repression in both stages, it would be reasonable for the parasite to use the same complex(es) to be effectively transmitted and establish infection in both directions. However, because the parasite will encounter and respond to significantly different conditions in these two events, it is also plausible that stage-specific proteins or complexes will be required. To

53 determine if Plasmodium parasites use stage-specific and/or stage-independent protein complexes, the first IP/MS of a cytosolic protein complex from Plasmodium sporozoites was performed. Historically, these experiments have failed due to the technical limitations and difficulties associated with producing and purifying large numbers of minimally perturbed sporozoites. Sporozoites must be manually dissected from mosquitoes, and soluble and insoluble material carried along from the mosquito (predominantly proteins, nucleic acids, and lipids) is in extreme excess compared to that of the parasite. This situation produces weak signal-to-noise ratios and other logistical blocks for many assays. For these reasons, most of the studies of translational repression in Plasmodium have focused on gametocytes. This experimentation has been able to overcome the obstacles of conducting these experiments with sporozoites by using a discontinuous gradient centrifugation, which is scalable and produces well-purified and fully infectious sporozoites from both human-infectious (P. falciparum, P. vivax) and rodent-infectious (P. yoelii) species [143]. This approach has allowed for unprecedented total and surface proteomics and transcriptomics of sporozoites [106] and is now used to conduct the first IP/MS experiment with protein complexes in sporozoites. Due to the pyalba4- oocyst sporozoites remaining associated with the midgut instead of invading the salivary gland (Figure 5B), this stage was selected for experimentation. It is hypothesized defects in this stage are responsible for this phenotype and determining PyALBA4’s binding partners may elucidate its function at this stage. To identify proteins associating with PyALBA4 in the oocyst sporozoites, immunoprecipitations from five million purified WT-GFP and PyALBA4::GFP oocyst sporozoites were conducted in biological duplicate. This procedure was largely the same as described above, however formaldehyde cross-linking was not possible with these samples as the crosslink reversal conditions necessary to break the formaldehyde bonds were too harsh for such delicate material. Therefore, binding and washing conditions were relaxed so that protein:protein interactions would be preserved. Due to this, transient and weakly interacting proteins were unlikely to be maintained, but still allowed identification of proteins with the strongest and/or most direct interactions with PyALBA4 (Figure 15).

54

Figure 15. Immunoprecipitation from oocyst sporozoites is feasible with purification and affinity-based techniques. Parasite lysates from 5 million purified oocyst sporozoites from either WT-GFP or ALBA4::GFP parasites were treated as above, except they were not chemically cross-linked. The full length ALBA4::GFP protein was recovered in the elution, as well as N- and C- terminally proteolyzed species (indicated by arrows). Based on approximate band sizes, it is predicted cleavage is occurring within the ALBA domain, and perhaps the GFP and the C- terminus is being cleaved off. In sporozoites, PyALBA4 is still associated with other ALBA proteins, specifically PyALBA1 and PyALBA2 (Table 4). However, PyALBA3 is conspicuously absent, despite it being detected at similar levels as PyALBA1 and PyALBA2 in schizonts and gametocytes. PyALBA3’s absence may be for two reasons: (1) PyALBA3 does not directly interact with PyALBA4, or (2) the interaction between PyALBA3 and PyALBA4 is weak or transient, requiring additional proteins or RNA for stabilization. Interestingly, four other proteins were identified though they were each only present in a single replicate. Two of these proteins are conserved proteins with unknown functions (PY17X_1002400, PY17X_0409300). Another (PY17X_1002400) was also detected in the schizont dataset, although below the stringent threshold used. In addition, in sporozoites (but not in gametocytes or schizonts) PyALBA4 associates with a predicted BRIX domain- containing protein (PY17X_0711900). Together, this raises the possibility PyALBA4 may associate with proteins both in a stage-independent (PyALBA1, PyALBA2, PY17X_1002400) and in a stage-specific manner (PY17X_1002400, BRIX). In other species, proteins containing this BRIX domain (e.g. Peter Pan, SSF1, SSF2) are important; in Xenopus these proteins are critical in the maternal cellular lineage and implicated in cell growth and division in yeast. These proteins are also implicated in ribosome biogenesis and rRNA binding [176-178]. This association is consistent with other PyALBA4 interactors identified in schizonts (described in Chapter 4), where PyALBA4 is also associated with ribosomal subunits and ribosomal biogenesis factors. This may indicate PyALBA4 is again acting as a broad mRNA interactor in oocyst sporozoites as well. However, the technical limitation of being unable to formaldehyde cross-link these complexes in sporozoites currently prevents the identification of other PyALBA4-binding

55 partners and thus further studies are warranted to gain a more complete understanding of PyALBA4’s role in this stage.

PyALBA4 affects transcripts encoding RNA-binding proteins in oocyst sporozoites Finally, the oocyst sporozoite was interrogated with the first reported comparative RNA-seq analysis of this stage in order to elucidate the function of PyALBA4 and understand why its absence results in the loss of synchronous development of sporozoites (Figure 5B). While PyALBA4 plays a role in mRNA turnover in schizonts and a protective and translationally repressive role in gametocytes, data from oocyst sporozoites indicates it may participate in both roles in this stage (Figure 16). These experiments are technically challenging (e.g. low amounts of material, persistent mosquito contamination), and thus two biological replicates were performed. The threshold for notable changes in transcript abundance was a >2-fold increase or decrease. Using this cutoff, 1131 transcripts exhibited differences in abundance in pyalba4- parasites (Table 11*). As seen in other stages, a large number (446) of transcripts encode for conserved unknown protein products with potentially important functions in sporozoites, but due to a lack of information on their predicted function they were excluded from this analysis. The remaining transcripts affected in this stage include a more balanced number of increased and decreased abundances (Figure 16C) and are distinct from the other stages interrogated (Figure 16D). Separate GO term analyses were performed on transcripts with abundance increases and decreases to understand what effect PyALBA4 may have during this stage (Tables 12, 13). First, focusing on transcript abundance increases, two notable GO terms were identified: RNA-binding and translation factor activity. Transcripts falling into these GO terms include transcripts encoding for eukaryotic translation initiation factors (e.g., EIF4G, EIF3C, EIF3E; see Table 11* for a full list). There are also six different DEAD/DEAH box helicases, and six RNA-binding proteins, some with bioinformatically inferred functions in splicing [132]. SRSf4, another splicing factor, also exhibits a transcript abundance increase. There are also transcripts encoding protein members of stress granules, including MCM2, MCM3, and MCM9 [47]. Taken together these transcripts largely encode for classic stress granule proteins or for

56 proteins involved in translation, which may indicate PyALBA4 also functions in translationally repressive activities in the oocyst sporozoite stage.

Figure 16. PyALBA4 affects transcript levels in both stage-specific and stage-independent manners. Comparative total RNA-seq was performed with WT-GFP and pyalba4- parasite lines from purified gametocytes (A), schizonts (B), and oocyst sporozoites (C). Transcript abundance increases (green) and decreases (red) greater than a 2-fold change (dotted horizontal lines, y- axis) are indicated with genes arranged by their current gene ID number (PlasmoDB, v27) on the x-axis. Arrows indicate pyalba4 transcript. Three (schizont, gametocyte) or two (oocyst sporozoite) biological replicates were performed. A) In gametocytes, transcript abundance decreases included transcripts transcribed early in gametocytes and those encoding for key proteins in ookinete development. B) In schizonts, transcript abundance increases included transcripts encoding for proteins important for invasion and cytoskeletal processes. C) In oocyst sporozoites there are many more transcripts with differential expression, with transcripts encoding RNA-binding proteins overrepresented. D) Across the stages investigated, there are very few transcripts affected in multiple stages, indicating PyALBA4 Other notable transcript abundance increases interacts with mRNAs in a stage-independent and a stage-specific manner. suggest a PyALBA4 absence may lead to a mistiming of expression. A surprising increase in transcript abundance supporting this idea is a 4.4-fold change in the p28 transcript; p28 plays a critical role on the ookinete surface, but does not have a known role in sporozoites. Additionally, three members of the ApiAP2 family of transcription factors also exhibit transcript abundance increases (PY17X_1235000, PY17X_1405400, and PY17X_1456200). PY17X_1405400 transcripts were the most affected (~3- fold increase), and Py17X_1456200 transcripts are also affected in the absence of PyPuf2; these findings suggest PyALBA4 function and transcriptional regulation may be linked. The most robust GO term associated with transcript abundance decreases was heat shock protein binding (Table 14*). Some of these members include DNAJ and DNAJ-like proteins

57

(PY17X_1350100, PY17X_1241400, PY17X_1008400) which are chaperones, and therefore may participate in stress granules. Transcripts encoding other stress granule proteins also exhibit transcript abundance decreases. These include MCM6, EIF5A, CELF2, and CCT4. However, it is intriguing to note some transcript abundances encoding for stress granule proteins increase while others decrease. Considering this, it is possible translational repression machinery transcripts are also exhibiting both increases and decreases in abundance. Finally, a decrease in transcripts of CPW-WPC family proteins was detected, similar to their decreases seen in gametocytes. These proteins may serve a yet-to-be elucidated function in both stages, which may contribute to the phenotype(s) observed.

58

Discussion PyALBA4 is likely associating with a canonical stress granule in gametocytes In the absence of PyALBA4, two major phenotypes are observed: (1) the increase in the number of exflagellating males, and (2) a loss of synchronous development in sporozoites

(Figure 5). A similar exflagellation phenotype was observed in pbpuf2- parasites [98], though in P. falciparum the genetic deletion of Puf2 did not result in altered exflagellation numbers [127]. However, the similarity of this phenotype in another rodent malaria species in the absence of a RNA-binding protein, and likely translational repressor, indicates PyALBA4 may serve a similar function in gametocytes. In this stage it is hypothesized the increase in the number of activated male gametes is due to PyALBA4 acting as a translational repressor, and in its absence transcripts are dysregulated and are more readily susceptible to translation. Therefore, when mature male gametocytes are exposed to a temperature change, one of the major external stimuli triggering activation, activation happens more readily. Interestingly, no gross morphological changes were observed in the female gametocytes, though it is possible a more subtle phenotype was not observed. In female gametocytes, PyALBA4 is known to interact with translationally repressive complexes, namely the DOZI and CITH complexes. Based on PyALBA4’s expression pattern and other protein binding partners detected, it is proposed translational repression is its main contribution at this stage [116]. The expression pattern of PyALBA4::GFP is also consistent with this model. In both male and female gametocytes, expression is largely diffuse, but includes foci of greater intensity and nuclear adjacent expression (Figure 10B). The foci of greater intensity appear to overlap with DOZI in female gametocytes, which is expected given the known interaction between the two proteins in this study and previous work [107]. These complexes include many RNA-binding proteins, including the other ALBA proteins, PABP, CITH, eIF4E, eIF4A, Musashi, Bruno/CELF2, and GBP2. Interestingly, there were several other protein associations indicative of canonical stress granules, including EF1α, EF1β, and EF2, and cytoskeletal elements important for the translocation of stress granules [47]. Several splicing factors were also found to associate with PyALBA4 and DOZI and CITH complexes. As splicing factors are known in some cases to remain associated with newly exported mRNAs, it is

59 possible PyALBA4 is associating with transcripts as or just after they are exported from the nucleus, consistent with PyALBA4’s nuclear adjacent localization. As PyALBA4 is a RNA-binding protein and associates with a translationally repressive complex in gametocytes, differences in mRNA abundances in its absence were expected. The technical difficulties of working with multiple stages and high host contamination were mitigated by treatment of infected mice with sulfadiazine to effectively kill the asexual population, and these gametocytes were captured by gentle centrifugation over a discontinuous gradient. Contamination from host material was still present, unfortunately limiting analyses to changes in only the most highly abundant transcripts, as these were likely preferentially sequenced. However, even with this limitation, clear effects of the absence of pyalba4 were observed. The changes seen are consistent with PyALBA4 acting as a translational repressor to protect transcripts. In PyALBA4 knockout lines, transcripts that encode for critical ookinete-related proteins exhibit transcript abundance decreases, including LCCL and CPW- WPC-domain containing proteins, PSOPs, GAP45, GAPM2, and WARP. There is no gross phenotype observed immediately after transmission into the mosquito vector, as determined by prevalence of infected mosquitoes and oocyst numbers; it is quite possible ALBA proteins have some redundant functions, or that the parasite is able to overcome some limited transcript abundance changes to an extent. It is possible the parasite may use translational repression of transcription factors to regulate gene expression and modulate mRNA homeostasis. Supporting this hypothesis, a decrease in transcript abundance of PY17X_1317000, an ApiAP2 transcriptional factor, is observed. This transcript and protein are of particular interest as it also associates with DOZI and CITH complexes [113]. Finally, two transcripts exhibit transcript abundance increases, and these are calcium-binding proteins. As other calcium binding proteins have been characterized and shown to be important in the activation of male gametocytes, the transcript abundance increases of these may explain the exflagellation phenotype observed. This suggests altered mRNA levels, and possible changes in mRNA stability and/or mistiming of protein expression are the reasons for the observed exflagellation phenotype. Therefore, PyALBA4’s main role in

60 gametocytes is tied to translational repression and the protection of specific transcripts that are important for the next stage of the life cycle. Additionally, in pyalba4- gametocytes, Nob1 is a downregulated transcript, indicating PyALBA4 may serve an important function in the quality control of ribosomes, typically by positively regulating Nob1. Its decrease in transcript abundance, and therefore potential decrease in protein and quality control may be what allows such a high number of gametocytes to activate. This reduced barrier to activation may lead to activation at the wrong time. It is possible this inadvertently allows an advantage to parasites, as they go on to produce viable, albeit not necessarily the most fit, parasites in the mosquito. PyALBA4 likely has a quick and transient interaction with Nob1 protein, which may explain why it is not immunoprecipitated with Nob1, though in its absence Nob1 transcript regulation is dysregulated.

PyALBA4 plays an as-yet defined role in sporozoites Based on comparative RNA-seq data, it is hypothesized that PyALBA4 associates with both a translationally repressive complexes and mRNA decay complexes in sporozoites. This is consistent with the localization of PyALBA4, as it is both cytosolic, punctate, and also nuclear adjacent in oocyst sporozoite stages. The expression pattern is reminiscent of PyPuf2 expression in salivary gland sporozoites, which also appears cytosolic and punctate [106]. It appears there is a mixed population of sporozoites, some of which are still fully infectious and successfully reach the salivary gland, though a majority remain developmentally stunted. This is likely due to two factors: 1) altered mRNA homeostasis in the absence of PyALBA4, which prevents proper sporozoite development, and 2) possible redundant function of the ALBA proteins that allow for a subset of sporozoites to egress from the midgut, invade the salivary gland, and remain infectious. The association of PyALBA4 with ALBA proteins and other RNA- binding proteins suggests PyALBA4 is participating in a conserved mechanism of translational repression preceding and during transmission events, though this process may be achieved by different proteins. This is also consistent with the differential transcript abundances noted in the pyalba4- parasite, wherein transcripts encoding for different proteins involved in stress granules exhibit either transcript abundance increases or decreases. It is currently unclear why

61 transcripts are affected in both ways. Additionally, the differential regulation of heat shock proteins is notable, as it is thought that these are important for stress granule movement and/or remodeling of these complexes.

62

CHAPTER 4 THE ROLE OF TRANSLATIONAL REPRESSION IN ASEXUAL DEVELOPMENT OF RODENT MALARIA PARASITES Results pyalba4- parasites exhibit no defects in asexual or liver stages While there were notable phenotypes observed in transmitted forms of the parasite, in asexual blood stages there were no major defects. Both clonal lines were tested, and the increase in parasitemia and parasite clearance of pyalba4- parasites were similar to those parameters observed for a wild type parasite line expressing GFPmut2 from a dispensable locus (WT-GFP) (Figure 17). In order to investigate PyALBA4’s function in asexual stages, the PyALBA4::GFP line described above was used. No morphological or behavioral defects with this line were observed compared to WT-GFP. PyALBA4::GFP is expressed throughout the blood stage cycle; in the asexual ring stage, its localization is nuclear adjacent foci. In trophozoites and schizonts, PyALBA4 remains punctate in cytoplasmic foci and near the parasite plasma membrane (Figure 10A). This is consistent with expression and localization profile of PfALBA4 in P. falciparum asexual blood stages [131] and several of its previously described binding partners [181, 182].

Figure 17. pyalba4- parasites do not exhibit any defect in blood stage growth kinetics. 1,000 blood stage parasites were IV injected into naive SW mice and daily parasitemia checks were done by thin blood smear followed by Giemsa stain. There were no significant differences in parasitemia, peak parasitemia, or timing of clearance. Two biological replicates are shown, represented as the average with standard error around the mean.

No gross phenotype in the pyalba4- parasites was observed during liver stage development, however PyALBA4::GFP is expressed in mid (LS24), late (LS48), and very late (LS52) liver stage (Figure 18). PyALBA4::GFP localization is diffuse and cytoplasmic during mid- liver stage, however, during late and very late liver stages the expression pattern changes to large foci, and then to small foci (Table 17). These expression patterns match the final steps of liver stage development as the parasites are segregated and packaged into exoerythrocytic

63 merozoites for hepatocyte release. This packaged, cytoplasmic diffuse PyALBA4::GFP may provide continuing functions for the parasite as it initiates the blood stage of infection.

Figure 18. PyALBA4::GFP is expressed throughout mid- to very late liver stages, and appears to be packaged into daughter merozoites. A) In liver stage, expression progresses from diffuse in mid-liver stages (LS 24 hr) to punctate in late and very-late liver stages (LS 48 and LS 52 hr). Mid-liver stage PyALBA4::GFP expression is solely diffuse, however in late and very-late liver stages, PyALBA4::GFP localization is restricted to the forming daughter merozoites. 100 parasites were assessed at each time point. Scale bars are ten microns.

PyALBA4 plays a multi-faceted role in blood stage schizonts Despite the lack of any defects in the pyalba4- parasites at this stage, the prevailing hypothesis is PyALBA4 plays a more general role in mRNA homeostasis in schizonts. This is consistent with its interactions with proteins involved in nuclear-to-cytoplasmic transport, canonical stress granules, active translational machinery, and some proteins involved in mRNA and protein decay/degradation (Tables 15, 16). It is important to note P. berghei and P. yoelii schizont samples (including these), though highly enriched, do also contain 10-15% gametocytes [183]. Consistently, overlap in the composition of the PyALBA4 complexes is seen between gametocytes and schizonts (Figure 13B), which is likely due to the presence of gametocytes, but may also reflect the same interactions occur in both stages. The 28 of 33 proteins found in both gametocyte and schizont samples include 19 members of the DOZI/CITH

64 complexes. Of the 133 proteins associated with PyALBA4 in schizonts, 88 are specifically associated with PyALBA4. As expected, PyALBA4 complexes isolated from schizonts share 44 proteins with the DOZI/CITH complexes, including DOZI, CITH, Musashi, CELF2/Bruno, PABP1, eIF4E, and others (Figure 13C; Table 15*). In this stage, more proteins found in the DOZI, CITH, and PyALBA4 complexes overlap than in gametocytes samples, including small and large ribosomal subunits and cytoskeletal proteins such as alpha tubulin II and a putative tubulin beta protein. In addition to its functions in storage granules, there is greater evidence PyALBA4 may be associating with newly exported mRNAs. In schizonts, PyALBA4 associates with far more proteins involved in nuclear-to-cytoplasmic shuttling than detected in gametocyte samples. RNA helicases, splicing factors, and other proteins involved in mRNA export were detected (Tables 15, 16). Two of these RNA helicases are DEAD/H Box helicase 5 (DDX5) and UAP56. DDX5 localizes to the nucleus in other species, and UAP56 is a known splicing factor with a structural fold similar to eIF4A which localizes largely in cytoplasm of schizonts [184, 185]. Proteins involved in mRNA splicing are also detected, such as the CELF family, SR1, Tudor Staph Nuclease (TSN), and a putative asparagine-rich antigen that in T. brucei associates with the cis- splicosome [186]. In addition, a conserved unknown protein (PY17X_0507200) containing a PHAX domain, typically found in proteins implicated in RNA export and ribosome biogenesis, is present [187]. Interestingly, PABP2 is also detected, which localizes to a nuclear and/or nuclear adjacent position in asexual blood stages. PABP2 is a known NPG protein component in Trypanosomes [65]. Because these proteins associate with PyALBA4 in schizonts, and the only location they coexist is adjacent to the nucleus or at the nuclear pore complex, a model for ALBA proteins interacting with newly exported mRNAs and/or a NPG is plausible (Figure 19). These findings are highly consistent with canonical stress granules. Excitingly, in further support of PyALBA4 interacting with exported mRNAs, interactions with casein kinase 1 and 2α (CK1, CK2α) were observed. CK1 and CK2 are serine/threonine kinases, and are involved in many processes including nuclear/cytosolic shuttling [181]. CK2α has been shown to directly phosphorylate ALBA1 and ALBA2 in vitro, and can be found in both the nucleus and in cytoplasmic foci resembling the ALBA4 expression pattern in schizonts.

65

Previous IP/MS experiments of CK2β identified 22 proteins that are also identified by PyALBA4 IP/MS in schizonts. The proteins detected that are specific to PyALBA4 belong to stress granules (Table 18*). This further supports the hypothesis that PyALBA4 is interacting with mRNAs either within or directly adjacent to the nucleus as they are exported. PyALBA4’s interactions with active translational machinery further support the hypothesis that PyALBA4 acts to regulate mRNA homeostasis. In schizonts, PyALBA4 associates with both small and large subunit ribosomal proteins. This is in stark contrast to the PyALBA4 complex in gametocytes, where only small ribosomal subunits are present, as would be expected from model eukaryotes if PyALBA4 is contributing to the translational repression of these mRNAs. Further evidence that PyALBA4 may be involved in active translation in schizonts comes from its association with elongation factors (EF1alpha, EF1beta, EF1gamma, EF2), eukaryotic initiation factors (eIF2g, eIF3E, eIF3D, eIF3G, and eIF5A), and Poly-A interacting protein 1 (PAIP1), which stimulates translation by interacting with eIF3 subunits involved in active translation (Tables 15, 16). Although PyALBA4 does not associate with many defined P-body specific proteins [132], some notable proteins from this complex are detected, including DOZI, CITH, eIF4E, and PABP2. As noted previously, homologs in model eukaryotes of DOZI (DDX6, Dhh1), CITH (Lsm14A, Scd6), and eIF4E are present in both stress granules and P-bodies [47]. PyALBA4 also associates with PABP2, a NPG protein, and is likely involved in mRNA degradation. Enhancer of rudimentary, a protein demonstrated to promote mRNA decay in the context of meiosis, was also detected [188]. While these associations strongly suggest PyALBA4 is playing a role in regulating mRNA homeostasis through mRNA degradation, the canonical members of P-bodies, such as members of the CCR4-NOT complex and DCP1/2, are not detectable associated with PyALBA4, perhaps due to the stringent statistical cutoff used here. It will be important to determine if the models of recruitment and interaction between granule types proposed for model species also apply to Plasmodium.

Table 16. PyALBA4 Interactions of Note in Schizonts. Gene ID Product Description # Spectra AvgP PY17X_1366000 ALBA4 138|213|230 -- Stress granule components

66

PY17X_1441700 PABP 98|38|78 1 PY17X_1208200 GBP2* 34|34|35 1 PY17X_1[400 conserved Plasmodium protien, unknown function 53|13|64 1 PY17X_1220900 DOZI 52|15|36 1 PY17X_1364900 ALBA2 72|26|32 1 PY17X_1035100 Bruno/HoBo/CELF2* 44|14|35 1 PY17X_0705000 conserved Plasmodium protien, unknown function 36|17|29 1 PY17X_1318600 EF2 52|8|31 1 PY17X_1304900 CITH 55|18|29 1 PY17X_0524100 alpha tubulin 2 16|27|45 0.99 PY17X_0415700 eIF4E 17|16|22 0.99 PY17X_1357200 EF1gamma 16|13|42 0.99 PY17X_1425300 ALBA1 71|47|23 0.99 PY17X_1119700 CDC48 12|3|13 0.99 PY17X_0512000 40S ribosomal protein S2, putative (RPS2) 12|9|11 0.98 PY17X_0822200 HSP70-2 15|6|12 0.98 PY17X_0821000 Musashi, HoMu 12|14|18 0.98 PY17X_0817500 EF1beta 7|6|16 0.98 PY17X_0407400 CCT2 5|2|8 0.98 PY17X_0521000 conserved Plasmodium protien, unknown function 8|4|4 0.98 PY17X_1437600 conserved Plasmodium protien, unknown function 5|5|7 0.97 PY17X_1361400 MyoA 3|3|3 0.96 PY17X_03[00 CCT8 5|1|5 0.92 PY17X_1207600 ALBA3 31|57|40 0.74 PY17X_1210100 tubulin, beta chain 40|31|69 0.72 PY17X_1134900 EF1alpha 142|47|13 0.62 PY17X_1446600 CCT3 11|0|3 0.6 Active translation machinery PY17X_0942100 PAIP1 42|9|18 1 PY17X_0407800 60S ribosomal protein L7 19|8|16 0.99 PY17X_0605700 eIF5A 3|2|4 0.94 PY17X_0715400 eIF3G 4|1|4 0.92 PY17X_1246200 eIF3E 9|1|5 0.9 PY17X_1209300 eIF3D 10|1|12 0.86 PY17X_1034300 eIF2gamma 1|2|11 0.74 mRNA export PY17X_1213400 PREBP 61|20|54 1 PY17X_1035100 Bruno/HoBo/CELF2* 44|14|35 1 PY17X_1242000 Karyopherin beta 13|20|38 1 PY17X_0524100 alpha tubulin 16|27|45 0.99 PY17X_0939200 asparagine-rich antigen, putative 30|7|10 0.99 PY17X_0507200 conserved Plasmodium protein, unknown function 18|3|11 0.99

67

PY17X_1137200 CELF1* 13|5|9 0.98 PY17X_0407400 CCT2 5|2|8 0.98 PY17X_1313500 DDX5 12|5|10 0.98 PY17X_1453200 CELF4* 9|14|11 0.98 PY17X_0913800 TSN 17|2|6 0.97 PY17X_1235500 SR1 6|7|5 0.97 PY17X_0917700 TCP1 5|2|9 0.96 PY17X_0941100 CK2alpha 4|4|7 0.96 PY17X_0311400 CCT8 5|1|5 0.92 PY17X_0707700 CELF3* 12|1|7 0.88 PY17X_1210100 tubulin beta chain 40|31|69 0.72 PY17X_0913600 CK1 4|0|12 0.63 PY17X_0307400 UAP56 3|0|10 0.6 PY17X_1446600 CCT3 11|0|3 0.6 NPG/P-body components PY17X_0828100 PABP2* 11|2|4 0.96 PY17X_0521700 enhancer of rudimentary homolog 3|6|7 0.95 Phenotype-related components PY17X_0617900 CDPK4 9|2|11 0.98 PY17X_0619400 NEK4 3|4|4 0.94 Housekeeping/Unknown function PY17X_0410900 phosphoglycerate mutase 69|24|53 1 PY17X_1217500 ENO 50|38|67 1 PY17X_0806800 conserved Plasmodium protein, unknown function 42|12|13 1 PY17X_0104900 conserved Plasmodium protein, unknown function 17|6|8 0.99 PY17X_1217900 conserved Plasmodium protein, unknown function 22|14|22 0.99 PY17X_1144100 conserved Plasmodium protein, unknown function 21|5|10 0.99 PY17X_1330200 GAPDH 57|42|74 0.98 PY17X_1117900 conserved Plasmodium protein, unknown function 15|11|13 0.98 PY17X_1118800 MDH 7|6|10 0.97 PY17X_1127000 pyruvate kinase 37|3|28 0.95 PY17X_1426200 conserved Plasmodium protein, unknown function 2|5|10 0.89 PY17X_1426300 conserved Plasmodium protein, unknown function 5|1|1 0.82 PY17X_1312400 ALDO2 36|36|53 0.77 PY17X_1139200 conserved Plasmodium protein, unknown function 20|1|43 0.63 PY17X_0109000 OAT 4|0|8 0.6

68

Figure 19. Model of PyALBA4 roles in asexual and transmitted forms. PyALBA4 plays a stage-specific role in schizonts, gametocytes, and oocyst sporozoites. In schizonts, PyALBA4 binds with mRNAs in a sequence-independent way and may act as a sorting mechanism. This may occur through a NPG, or independently through interactions with mRNA export machinery. PyALBA4 does also interact with mRNAs involved in various fates, including active translation and translational repression, however the latter is not the dominant interaction in schizonts. In gametocytes, we propose that PyALBA4 is predominantly associating with translationally repressive machinery, though there is some low level detection of mRNA export machinery. Proteins associated with PyALBA4 are post-translationally modified As mentioned previously, no acetylation events in the unenriched peptides were detected, though it is unclear if acetylated proteins are not present or simply not detected due to lack of enrichment. While the phosphorylation findings in gametocytes and sporozoites were sparse, in schizonts post-translational modifications were readily detected on several notable proteins. Phosphorylation was detected on Bruno/CELF2 (residue S83 or S84) and PyALBA3 (S3 or T4) – the same modifications seen in gametocytes. GBP2, however, was instead phosphorylated at residue S7 and/or T9. Identification of phosphorylation events on EF1g

69

(S233), eIF3A (S1245), and PyALBA4 (S13) were also seen. Post-translational modifications, such as phosphorylation, can be important for binding to and determining the fate of mRNAs. As PyALBA4 associates with many different complexes in schizonts, it is possible the post- translational status of PyALBA4 helps to direct the bound mRNA to its appropriate location. This requires further experimentation.

PyALBA4 affects transcripts involved in invasion in schizonts A comparison of the average transcript levels between WT and knockout parasite lines in schizonts (Figure 16B) indicates PyALBA4 may predominantly participate in mRNA turnover functions, as most differences observed were transcript abundance increases and very few transcript abundances decreases in the pyalba4- parasite line (Figure 16B, Table 14*). Proteins exhibiting a statistically significant decrease were mostly YIR proteins, which are known to be clonally variant, and were thus removed from analyses [189]. As a result, 251 transcripts with statistically significant increases in transcript abundances were included in the analyses (Figure 16B, Table 14*). A GO term analysis on these data revealed transcripts involved in microtubule motor activity, microtubule binding, cytoskeletal protein binding, and tubulin binding showed increases in transcript abundances (Table 19). Transcripts fitting these GO terms included transcripts encoding several dynein heavy and light chain proteins, actin-related proteins (ADF2, ARC40), tubulins, as well as kinesins (e.g., kinesin-8). An increase in EB1 homolog (PY17X_0407900), which is responsible for recruiting kinesins to the microtubule in model eukaryotes was also detected [190]. Based on these findings, it is highly likely PyALBA4 interacts with certain transcripts known to affect invasion related processes. This finding is consistent with transcript abundance changes that are affected with PfALBA1 overexpression [114]. As late schizonts are preparing to egress and invade new RBCs, the proteins encoded by these transcripts are critical for successful propagation of blood stage. It is also possible PyALBA4 may be segregating these transcripts into daughter merozoites prior to RBC rupture, which is consistent with the PyALBA4 localization pattern being both cytoplasmic and nuclear adjacent in blood stage parasites; this pattern also appears to be packaged into exoerythrocytic merozoites (Figure 10A, 18).

70

Table 19. GO Term Analysis of transcript abundance increases in pyalba4- schizonts Genes % Genes in in background Fold Odds P- GO ID GO Term background result in result enrichment ratio value Benjamini Bonferroni Molecular Function 1.02E- GO:0003774 motor activity 24 9 37.5 13.54 15.42 07 1.47E-06 1.53E-06 microtubule motor 1.96E- GO:0003777 activity 18 8 44.4 16.05 18.02 07 1.47E-06 2.94E-06 cytoskeletal protein 6.26E- GO:0008092 binding 22 5 22.7 8.2 8.77 04 2.65E-03 9.39E-03 8.83E- GO:0015631 tubulin binding 13 4 30.8 11.11 11.73 04 2.65E-03 1.32E-02 8.83E- GO:0008017 microtubule binding 13 4 30.8 11.11 11.73 04 2.65E-03 1.32E-02 Cellular Component 5.81E- GO:0005856 cytoskeleton 53 11 20.8 7.49 8.72 07 5.81E-06 5.81E-06 microtubule associated 1.50E- GO:0005875 complex 25 8 32 11.55 12.94 06 6.73E-06 1.50E-05 2.20E- GO:0015630 microtubule cytoskeleton 37 9 24.3 8.78 9.95 06 6.73E-06 2.20E-05 2.69E- GO:0044430 cytoskeletal part 50 10 20 7.22 8.27 06 6.73E-06 2.69E-05 1.38E- GO:0030286 dynein complex 7 4 57.1 20.63 21.84 04 2.77E-04 1.38E-03 4.42E- GO:0005871 kinesin complex 10 3 30 10.83 11.28 03 7.36E-03 4.42E-02 Biological Process microtubule-based 9.86E- GO:0007017 process 33 10 30.3 10.94 12.62 08 5.88E-07 5.91E-07 microtubule-based 1.96E- GO:0007018 movement 18 8 44.4 16.05 18.02 07 5.88E-07 1.18E-06 cellular component 3.74E- GO:0006928 movement 20 8 40 14.44 16.2 07 7.47E-07 2.24E-06

In addition to these transcript abundance changes, increases in transcript abundance of transcripts encoding RNA-binding proteins were observed. These include NOB1, which is important for rRNA processing [191], Musashi, Puf1, and a currently uncharacterized RBP (PY17X_1457300). As PyALBA4 interacts with known translational repressors in this stage via IP/MS, it is hypothesized PyALBA4’s direct or indirect interaction with these transcripts may provide another level of regulation over proteins important for downstream translational regulation events (i.e., Musashi). In pyalba4- schizonts, Nob1, which is critical for ribosome maturation, transcript abundance is nearly doubled; this may mean the quality control function is less stringent at this stage, where proliferation is happening at a rapid pace and overall population is ramping up to prepare for the population bottleneck at mosquito entry.

71

Transcripts of proteins known to be important in male and female gametocytes and in ookinetes also exhibited differential expression. In particular, differences in transcripts of proteins involved in egress of male and female gametes were noted. These include a flagellar outer arm dynein-associated protein (PY17X_0505400), male developmental gene 1 (MDV1, PY17X_1434500), and gamete egress and sporozoite traversal protein (GEST, PY17X_1316500) [192-194]. In P. berghei it was demonstrated that MDV1 mediates both male and female gamete egress from the host RBCs [194]. Similarly, GEST is also known to be important for egress from host RBCs and is expressed in early gametocytes [193]. Transcripts encoding for proteins important in ookinetes with transcript abundance increases include CelTOS, PSOP2, and PSOP17. CelTOS transcript levels reach a peak level during the ookinete stage in P. berghei, and disruption of CelTOS results in decreased vector infectivity [195]. In ookinetes PSOP2 is implicated in successful infection of the mosquito, and in its absence, infection of mosquitoes is greatly reduced [196]. PSOP17 has not been fully characterized, but is hypothesized to play a similar role in ookinete biology [82]. Notably, these transcript abundances are increased in the absence of PyALBA4. Therefore, it is hypothesized there is some regulation of gametocyte and ookinete transcripts in late schizonts, specifically schizonts committed to becoming gametocytes; however, it is also possible that gametocyte contamination in the sample may contribute to these findings. Nonetheless, these transcripts encode proteins that may be linked to the phenotype observed. A comparison of these data to other studies of ALBA proteins may help elucidate PyALBA4’s function within its complexes. The data generated in the schizont stage data was compared to transcript abundance differences observed in the overexpression of PfALBA1, an essential blood stage gene (Figure 14B) [114]. Interestingly, transcripts belonging to similar GO terms are being affected between these datasets, including microtubule-binding and cytoskeletal components. However, most of the affected transcripts are different. This may be due to differences in approach (knockout vs. overexpression), species (P. yoelii vs. P. falciparum), or stage (schizonts vs. trophozoites). The schizont transcript changes dataset was also compared to a list of known translationally repressed transcripts, but again very little overlap was seen [197, 198] (Figure 14C). This observation lends further credence to the

72 proposed model that PyALBA4’s primary role in asexual stages is not to impose translational repression. It is also important to point out some of the most substantial changes in transcript abundances are seen in transcripts that encode for currently uncharacterized proteins (PY17X_0936800, PY17X_1248400). Further characterization of the products encoded by these transcripts may improve understanding of PyALBA4’s function and of Plasmodium blood stage biology.

Discussion PyALBA4 is involved in multiple processes regulating mRNA homeostasis in schizonts RNA-binding proteins have been characterized and are individually thought to be involved in many different aspects of mRNA metabolism. The hypothesis that a single RNA- binding protein can act in many, and possibly opposing, mechanisms in a single stage has recently gained momentum. Here, PyALBA4 is demonstrated to play both protective and degradative roles in many different aspects of mRNA homeostasis in a stage-specific manner. The appearance of cytosolic punctate foci in schizonts likely indicates PyALBA4, as a RNA- binding protein, is present in a mRNP. Coupled with the constant presence of nuclear adjacent localization, it is hypothesized PyALBA4 is acting in a multi-faceted way; PyALBA4 is interacting with a translationally repressive complex with mRNA, but may also be interacting with nascent mRNA transcripts as they are exported from the nucleus and then shuttled to the appropriate area for downstream processing. This could include active translation and/or mRNA degradation. The proteins associated with PyALBA4 in schizonts include proteins involved in translational repression, active translation, mRNA export, and mRNA degradation. As the production and maintenance of a preferred mRNA homeostasis is a dynamic process that must adjust as conditions change, it is likely the chemical cross-linking captures an in vivo snapshot of PyALBA4 in each of its various modes of action, which is a clear advantage of this approach. Though there are translationally repressive protein associations, such as DOZI and CITH and other members of these complexes (Figure 13C), there are many interactions specific to PyALBA4 complex(es). These include mRNA export and splicing factors, which can accompany

73 the mRNA as it is exported through the nuclear pore. As PyALBA4 is observed to be nuclear- adjacent, it is possible PyALBA4 is associating with these nascent mRNAs and their machinery. The association of PyALBA4 with NPG proteins members, such as PABP2, DOZI, and CITH also suggests PyALBA4 is involved in receiving and/or sorting mRNAs to their proper fate, which can include mRNA degradation. As NPGs often contain improperly spliced mRNAs in Trypanosomes targeted to degradation, this highlights PyALBA4’s possible role in degradation and quality control of transcripts. PyALBA4 also interacts with enhancer of rudimentary, which is another protein involved in mRNA degradation. While the canonical members of mRNA degradation complexes, such as CCR4-NOT complex members or DCP1/2 are detected in the experimentation, they do not fall above the stringent statistical thresholds. However, it is possible PyALBA4 is transiently or weakly interacting with this complex to transfer mRNAs targeted for degradation. The association with active translational machinery also indicates PyALBA4 may remain associated with mRNAs, and this interaction may prove important if a given mRNA needs to be shuttled into a translationally repressive or degradative complex, or rapidly brought back into the actively translated population of transcripts. Taken together, these observations suggest PyALBA4 may be playing a broad role by associating with mRNAs and helping to target them to their proper fate, whether that is active translation, translational repression, or degradation. The transcripts affected in the absence of pyalba4 indicate PyALBA4 is involved in regulating mRNAs that encode for proteins involved in invasion. These include cytoskeletal and mictrotubule-binding proteins, and this shares a striking similarity with the transcripts regulated by PfALBA1 [114]. However, while these proteins serve similar functions, the actual transcripts regulated by PfALBA1 differ from those in PyALBA4. This may due to the slight differences between experiments and datasets and inherent protein differences; PfALBA1 is essential, while PyALBA4 is not, therefore explorations of PfALBA1 employed an overexpression strategy instead of a genetic ablation. Additionally, the role of the PfALBA1 was explored in trophozoites, while this study of PyALBA4 focused on schizonts. However, Vembar and colleagues still suggest PfALBA1 is involved in translational repression, specifically of transcripts that encode proteins involved in invasion. This presents a reasonable explanation for the lack of

74 a gross phenotype in asexual blood stages in pyalba4- parasites: ALBA proteins may serve redundant and/or complementary functions throughout the parasite life cycle. This is also consistent with ALBA proteins associating with each other throughout the life cycle. Based on these observations, PyALBA4’s function in schizonts may also be important for packaging mRNAs properly prior to life cycle transitions, such as reinvasion of a new red blood cell. This is also consistent with the expression pattern observed in liver stage parasites, as it appears PyALBA4 is packaged into daughter merozoites prior to their release into the vasculature (Figure 18, Table 17). In this study, it was also possible to characterize the phosphorylation status of proteins involved in these complexes without the need to enrich for the modification. In yeast, the phosphorylation of a RNA-binding protein can affect the fate of its bound mRNA [199]. This was of particular interest, as post-translational modification of ALBA proteins is known to modulate their binding affinity to nucleic acids, and because ALBA proteins have been found in the P. falciparum phosphoproteome [200, 201], as well as oocyst- and salivary gland sporozoites stages (K. Swearingen, personal communication). In this study, it was possible to identify several post-translationally modified proteins that associate with the PyALBA4 complexes in gametocytes and schizonts (Table 8). These samples were not enriched for post-translational modifications, therefore this analysis is not comprehensive, but is encouraging, as these modifications are robust enough for identification. Notably, no acetylation modifications were detected, though several phosphorylated proteins, including PyALBA4 itself, were detected. This was only detected in the schizonts and not gametocytes, where translational repression prevails, suggesting the phosphorylation status of PyALBA4 may determine whether the mRNAs it associates with are translationally repressed. The association of PyALBA4 with proteins involved in many aspects of mRNA metabolism, including mRNA storage and repression, mRNA export, active translation, NPGs, and mRNA decay, across multiple stages underscores its importance. A plausible explanation for its interactions with a diverse group of proteins can be simply explained by a single interaction: mRNA. Therefore, a model where PyALBA4 indiscriminately binds mRNA in a sequence-independent manner, and potentially stabilizes these transcripts is proposed. In the

75 absence of a RGG domain or an FG-like domain, it is reasonable to assume PyALBA4 does not necessarily bind a specific set of transcripts, but rather interacts broadly with mRNAs. Due to PyALBA4’s nuclear adjacent expression and interactions with mRNA export proteins, it is plausible to conclude it is available to interact with nascent mRNAs and subsequent mRNPs. ALBA4 may remain associated with the transcripts regardless of its fate. It is possible the post- translational status of ALBA4 affects the fate of the bound transcript, either directly or indirectly by effector proteins. In stages where mRNA homeostasis is of tantamount importance, gametocytes and sporozoites, this is where the importance of PyALBA4’s action is most clearly seen. While the phenotypes observed in PyALBA4’s absence do not necessarily hinder the progression of parasite development or successful establishment of infection, they underscore the temporal importance of mRNA homeostasis. Taken together, these findings suggest PyALBA4 acts as a key regulator of mRNA homeostasis that may remain associated with the mRNA regardless of its fate (Figure 19). Several groups have demonstrated and reported evidence that at least two types of these discrete cytosolic granules are present in Plasmodium species: the DOZI/CITH complex and the PyPuf2 complex [106]. The presence of distinct granules is consistent with PyALBA4 datasets, where PyPuf2 is not shown to associate with PyALBA4 complexes, as well as with similar datasets from other groups who investigated DOZI and CITH complexes [98, 106, 115, 116, 127]. This is consistent with the observation that PyPuf2 granules do not co-localize with any existing antibody markers [106]. Therefore, the complexes PyALBA4 is associated with are distinct from the PyPuf2-containing complex and likely perform different, but complementary, functions for the parasite [116]. Based on these data and premises, a sequential mechanism for the release of translational repression in response to productive transmission is proposed. Parasites that are fully prepared for transmission have: (A) formed two types of translational repression complexes: one type acting as a specific translational repression complex (e.g. Puf2, DOZI/CITH/ALBA), and another acting as a globally repressive complex (e.g. resulting from the phosphorylation of eIF2α) [202, 203]. When the appropriate (but presently undefined) external cues are received (B), granules responsible for the specific translational repression

76 complex dissolve and allow the translation of their previously bound transcripts (C). However, the translation of these mRNAs would at first be limited in scale due to the global translational repression that is also in place, which would act to suppress translation (D). If one of these transcripts encodes the phosphatase(s) responsible for dephosphorylating eIF2α, the release of specific translational repression would then trigger the release of the global translational repression as well. It has been suggested UIS2 is this phosphatase [203]. The combined effect would allow the parasite to quickly respond to its post-transmission environment (e.g. mosquito midgut, hepatocyte) and translate the prepared mRNAs in a “just-in-time” manner. This model is attractive, as it provides several experimentally testable questions for both transmission events, such as 1) what is the external stimulus and how does the parasite sense it, 2) how is that response converted to ultimately cause granule dissolution, and 3) what is the phosphatase(s) responsible for release of global translational repression. One could imagine interventions in these crucial points may disable the transmitted parasite and disrupt the infection of the new host or vector.

77

CHAPTER 5 CONCLUSION AND FUTURE STUDIES Conclusions on PyALBA4 PyALBA4 plays a multi-faceted role in mRNA regulation In order to determine the roles played by PyALBA4 in mRNA homeostasis and to identify subsets of mRNAs it either directly or indirectly regulates, comparative total RNA-seq with WT-GFP and pyalba4- parasites at the gametocyte, schizont, and oocyst sporozoite stages were performed. As PyALBA4 localizes to areas where mRNAs are known to be regulated (e.g. adjacent to the nucleus, cytosolic mRNPs), it was hypothesized mRNA abundances may be affected by PyALBA4 during these stages and may offer insight to the phenotypes observed in pyalba4- parasites (Figure 5). First, as expected the pyalba4 transcript is among the most affected (Figure 16A-C) in the knockout line in each stage, indicating the gene was successfully disrupted. The percentage of reads mapped to the reference genome varies from stage to stage (Schizonts ~70-78%, Gametocytes ~2-10%, Oocyst sporozoites ~1-5%), but sufficient read depth allowed detection of changes in abundance in all stages with confidence. The analyses here have been limited to only the most abundantly expressed transcripts in those stages with lower mapped read percentages. Together these data show PyALBA4 affects mRNAs differently in these three stages (Figure 16A-C) and the mRNAs it regulates are largely stage specific (Figure 16D). Moreover, the data herein is consistent with and expands upon recent investigations of PfALBA1 and DOZI/CITH complexes [113, 114]. Taken together, the observed transcript abundance changes in gametocytes and oocyst sporozoites are congruent with and help to explain the observed phenotypes.

PyALBA4 regulates transcripts stage-specifically and stage-independently Taking these total comparative RNA-seq datasets together, it is clear PyALBA4 plays opposing and mixed roles in regulating distinct transcript types relevant to specific stages of development. Intriguingly, there is very little overlap between the differentially expressed

78 transcripts between stages (Figure 16, Table 20*). There are only six transcripts shared across all three life stages, and four of these encode for uncharacterized proteins. There is also very little overlap between gametocytes and schizonts, which is surprising given gametocytes are found in the enriched schizont samples. Regardless, it seems the schizont and gametocyte samples are distinct enough to disentangle PyALBA4’s function in each stage. Additionally, 21 of the 22 transcripts that do overlap encode for uncharacterized protein products. Finally, when comparing oocyst sporozoite transcript changes to either schizonts or gametocytes, again we see a distinct set of affected mRNAs, with only a few notable overlaps. One such transcript is CCp5, which increases in schizonts, yet decreases in oocyst sporozoites. However, most of the transcript abundance changes mentioned previously throughout these sections are stage- specific, indicating that PyALBA4’s role in directly and/or indirectly regulating these transcripts is both stage-specific and stage-independent, and may provide a conserved mechanism to achieve the desired mRNA balance across the life cycle.

Concluding remarks The association of PyALBA4 with proteins involved in many aspects of mRNA metabolism, including mRNA storage and repression, mRNA export, active translation, NPGs, and mRNA decay, across multiple life cycle stages underscores its importance. Therefore, a multi-step model is proposed. PyALBA4 indiscriminately binds mRNA in a sequence- independent yet stage-dependent manner, potentially (de)stabilizes these transcripts. In the absence of a RGG domain or an FG-like domain, it is reasonable to assume that ALBA4 interacts broadly with mRNAs, which could occur through interactions with the ribosome. Due to PyALBA4’s nuclear adjacent expression and interactions with mRNA export proteins, it is plausible that it is available to interact with nascent mRNAs and subsequent mRNPs, or in a quality control manner for ribosomes. PyALBA4 may remain associated with the transcripts regardless of its fate. It is possible that the post-translational status of PyALBA4 affects the fate of the bound transcript, either directly or indirectly by effector proteins. In stages where mRNA homeostasis is of tantamount importance, gametocytes and sporozoites, the importance of ALBA4’s action is most obvious. While the phenotypes observed in ALBA4’s absence do not

79 necessarily hinder the progression of parasite development or successful establishment of infection, they underscore the temporal importance of mRNA homeostasis. Therefore, PyALBA4 acts as a key regulator of mRNA homeostasis that may remain associated with the mRNA regardless of its fate. This study demonstrates how Plasmodium parasites can utilize a single regulatory protein differently across its life cycle, simply by modifying the composition of its protein complex. Presumably this is done to affect different mRNAs as needed for the successful growth and transmission of the parasite between the host and vector. I argue exploiting what we know about stress granules and their existence in transmitted forms of the parasite can elucidate a clear malaria control strategy. By drawing parallels to what is known about stress granule biology in neurodegenerative diseases, I think it is plausible to target the parasite. With the rampant spread of drug resistance, novel therapy treatments are badly needed. One way to exploit this basic biology is to engineer the introduction of “sticky peptides” into Plasmodium, which will lead to protein aggregation, and hopefully whole cell dysfunction, as seen in neurodegenerative diseases. Another avenue is blocking the clearance of these stress granules, by targeting autophagy-related proteins. In order for these strategies to be successful, understanding of discrete mRNP granules in Plasmodium must go deeper. To that end, I suggest the following experimentation.

Future Studies There are several experiments I believe will deepen understanding of the basic biology of stress granules and novel storage granules in Plasmodium. Given what is known about the nucleolus and its response to stress, an interesting first experiment is cryoEM to determine if there are any nucleolar size, shape, or number changes in pyalba4- parasites compared to WT-lines. This could be done at several life cycles points, especially parasites exposed to a mimicked transmission event, e.g., gametocytes exposed to a rapid reduction in temperature or altered pH environment. If there is a ribosomal stress, as hypothesized, the expectation would be nucleolar changes. To further characterize stress granules in Plasmodium, one of the most informative experimentation would include generating markers of canonical stress granule components in

80

Plasmodium. These are severely lacking in Plasmodium and generating these it is not a simple task; it would require either genomic manipulation to tag individual proteins, or the generation of anti-sera against each protein of interest. The most important markers include eIF2a, eIF2a- P, G3BP, and the possible TIA-1 homologue. It would also be advantageous to have distinct ALBA antibodies, to help understand how this family of proteins may be interacting with each other. In model organisms, it is well established stress granule composition is driven by the stressor applied to the cell. To deepen understanding of stress granule composition in Plasmodium, I suggest perturbing parasites with multiple classic stressors such as temperature, pH, nutrient deprivation, amino acid starvation, etc., and determining the resulting stress granule complexes through immunohistochemistry, protein immunoprecipitation, RNA immunoprecipitations. An interesting stressor to include for Plasmodium would be typical antimalarial treatment. Following collection and identification of the components in stress granules, comparing them to those performed with PyALBA4 above would create a granule- ome for Plasmodium and deepen the understanding of PyALBA4 and other proteins functions. Because ALBA4 is shown to associate with many different proteins with diverse functions and it is unclear which components correspond to which granules, I suggest employing subcellular fractionation of cell lysates to pin down how these complexes may be spatially arranged. This will also determine how important localization is to protein/granule function and tease apart which components associate with each granule type described above. On a very basic level, I firmly believe that the proteomics and transcriptomics data mentioned above should be validated by other experimental means, including qPCR, super-resolution and fluorescent microscopy to determine whether these proteins are actually associated with each other or are simply nearby. Coupling these experiments with subcellular fractions is a technically difficult experiment to perform, but would offer substantial experimental findings and aid in deepening understanding. Further, understanding how post-translational modifications affect stress granule formation in Plasmodium is also an interesting avenue of experimentation. Evidence of PyALBA4 phosphorylation begs the question – how does it affect stress granules if it is

81 constitutively phosphorylated? To take a step back – is PyALBA4 ever constitutively phosphorylated? To answer this question, I propose using a phosphomimetic version of PyALBA4. Additionally, we can ask if stress granules may persist if PyALBA4 cannot be phosphorylated. To do this, mutations on Serine 51 should be sufficient to block phosphorylation. Immunofluorescent assays, phenotyping, and immunoprecipitations using these mutated proteins would go a long way in answering some of these basic questions. A critical experiment I was not able to perform is RNA-immunoprecipitations with these ALBA4 complexes. This is preferable over the total RNAseq data, as the changes observed with RNA-immunoprecipitations would be directly due to PyALBA4 action. Even though PyALBA4 lacks a clear RNA-binding motif, creating the PyALBA4 RNA Interactome (ARI) would offer additional evidence of its role and function in different life cycle stages. Additionally, ribosomal footprinting may also elucidate what transcripts are preferentially translated at specific life cycle stages, and when preferential translation is occurring. With multiple life cycle stages, there is no dearth of interesting time points to interrogate. I believe this could even be done with sporozoites, given the purification technique employed in the above experiments. An interesting yet frustrating aspect of this project is the poorly annotated Plasmodium genome. Though ‘hypothetical unknown proteins’ were removed from the analyses performed above, it was not done because these proteins do not have merit. There are well over fifty proteins that have interesting domains that may be involved in stress granules. Characterizing these proteins with classic reverse genetic techniques is an arduous, but potentially very rewarding task. While those with clear domains are of interest, there may be greater value in characterizing proteins with no predicted domains that interact with the PyALBA4 complex, including PY17X_0806800 and PY17X_1117900. These are a black box which may illuminate other functions specific to PyALBA4 when it is not in a DCA complex. Beginning with proteins that associate with ALBA4 in gametocytes is a prime starting point, as this is consistent with wanting to exploit the most vulnerable stages at the points of transmission. Because there are often large disparities between what is seen in human-infectious and rodent-infectious parasites, exploring the role of ALBA4 in Plasmodium falciparum is also an

82 important avenue to consider. While generating knockouts in P. falciparum is classically more difficult and time consuming, the advent of CRISPR technologies has reduced the time commitment. P. falciparum sporozoite studies are also attractive, as they could be highly impactful. Additionally, any control strategy would necessarily need to be worked out in human-infectious species, so beginning there reduces time and increases impact. As discussed, stress granules need to be cleared, and it is possible the coacervates or aggregates left behind from improper clearance may lead to dysfunction. Targeting proteins such as CDC-48, which is important for autophagy, can begin to elucidate whether these aggregates cause dysfunction in Plasmodium. It is also unclear if autophagy is even involved in clearance of these granules. Generating a CDC-48 knockout line and CDC-48::GFP line would be useful in determining defects in autophagy specifically in the context of stress granules. Because post-translational modifications are hypothesized to play a role in targeting stress granules for disassembly, coupling the CDC-48 experimentation with ALBA4 phosphomimetc experiments would allow a deeper dive into the biology in Plasmodium. This leads to my final and favorite experiment. I hypothesize the introduction of a ‘sticky’ peptide or protein may cause aggregation, and in turn lead to whole cell dysfunction. This may even result in apoptosis. The introduction of this sticky peptide can be difficult, especially the targeted introduction of exogenous material. An alternate route is to trigger the overexpression of a protein that contains a prion-like domain or low complexity ‘sticky’ region, such as ALBA4, or Bruno. If stress granule formation is driven by liquid-liquid phase separation due to concentration thresholds, flooding the cells with a sticky protein increases the probability more stable complexes will form and remain, regardless of potential clearance efforts. With CRISPR technologies this has become a tractable experiment. By using a parasite line with integrated Cas-9, this experiment could even be done in a relatively short amount of time.

83

APPENDIX A Revisions to Chapter 3 The final manuscript form of my dissertation work differs in this particular section. I was unable to perform the immunoprecipitations of oocyst-sporozoites due to issues with cross-linking a delicate and minimal sample. In my attempts to reverse the cross-links, the entire sample was lost. This was not discovered until after I performed extensive troubleshooting, however I then had to depart from the Lindner lab. In my opinion, it was better from a tractable experimental standpoint to move forward without cross-linking the material. However, Dr. Lindner disagreed. He and Kevin Hart further pursued these experiments, and were able to successfully cross-link and immunoprecipitate a complex from oocyst-sporozoites. However, the cross-link reversal was not performed in these experiments. This in turn led to problematic and inconclusive downstream proteomics and bioinformatic analyses, as it is not possible to determine whether any of the inferred proteins are in fact correct identifications without extensive validation, which was not carried out. I have included their data in this appendix. “Because only a few interacting partners of ALBA4 were detected without crosslinking, we also pursued the same experiments but using formaldehyde to stabilize transient interactions through crosslinking, but using extensive washing in lieu of crosslinker quenching. Again, experiments with sporozoites were only feasible upon extensive purification with an Accudenz gradient as we have previously described. However, even starting with comparatively substantial numbers of sporozoites, these experiments yielded very little mass following immunoprecipitation compared to blood stage parasites. This is perhaps seen most readily by the resulting average total spectral counts for ALBA4::GFP for each sample type (Schizont: 90.7, Gametocyte: 193.7, and Oocyst Sporozoite: 37.7). Because of this, we again present the data using two statistical cutoffs. These cutoffs include the use of the same highly stringent and moderately stringent SAINT thresholds as with other samples (0.1 and 0.35), that similarly led to the inclusion of known interaction partners of ALBA4 from other stages. Complete datasets for all three stages with both thresholds noted are provided (Additional File 3, Tables S5, S7, and S10). In sporozoites using the highly stringent (0.1) threshold, we have identified several proteins in addition to ALBA4 that are in common in all three pulldowns, including core

84 components of the this complex from other stages. These include ALBA1, ALBA3, Musashi, GAPDH, HSP90, the ribosome, and PABP (Table 4). Upon expanding this threshold, many additional members of the DOZI/CITH/ALBA complex from blood stages are then included, such as ALBA2, Bruno/CELF2, GBP2, Aldolase, UAP56, and ribosomal proteins from both small and large subunits (Table 3).”

Table 21. Oligonucleotides used in this study

85

Table 22. Revised oocyst sporozoite table

86

APPENDIX B Contribution to “PlasmoSEP: Predicting surface-exposed proteins on the malaria parasite using semisupervised self-training and expert-annotated data” I contributed significant experimental work, writing, and intelligent discussion to the scientific paper titled, ““PlasmoSEP: Predicting surface-exposed proteins on the malaria parasite using semisupervised self-training and expert-annotated data” [205], for which I was granted second authorship. The paper details a new biostatistical framework, called PlasmoSEP, to utilize for improved identification of surface exposed proteins (SEPs) on the outside of the parasite membrane. The framework is a novel semisupervised learning algorithm which combines historically annotated SEPs identified by high‐throughput experiments and expert annotation of these datasets to provide a training dataset for a predictive model. After training the model, the learning algorithm was employed for validation purposes, wherein known SEPs found in Plasmodium falciparum not fed into the training dataset were identified. After the validation, the algorithm was employed to predict SEPs in Plasmodium yoelii. The algorithm correctly predicted and identified with high-confidence 25 of 37 experimentally identified SEPs in Plasmodium yoelii salivary gland sporozoites, which are likely to be SEPs. I performed the validation experimentation in Plasmodium yoelii to confirm the findings of the algorithm. Below is Figure 1 from the paper, detailing the overall workflow.

EM Major Contribution

87

APPENDIX C PUF2 KO Analysis To further explore the knockout phenotypes of puf2- parasites, I carried out exflagellation assays as described above. I compared a WT-GFP expressing line to two clonal KO lines, and performed three biological replicates for each parasite line. These parasites exhibited a trended decrease in exflagellation numbers. However, when compared to the WT-GFP line, neither clone was statistically significant (Student T-test, p-values >0.1). In addition to these experiments, I also generated total RNA-seq data from PyPuf2KO and CCR4KO parasites. Again, this was performed in triplicate for each parasite line, and a WT- GFP line was used as a control. The raw data was analyzed by Michael Walker, and a brief analysis was performed. Most hits of statistical significance were YIR transcripts, which are known to change expression rapidly. Other hits included some mRNAs involved in translational repression, but the dataset was not robust enough for meaningful conclusions due to the low read count and depth.

Figure 20. pypuf2- clones exhibit a trended decrease in exflagellation. Exflagellation assays as described above were carried out on two clonal pypuf2- parasite lines. A trended decrease in the number of centers of movement was observed, however the difference was not statistically significant.

88

APPENDIX D PUF2 Proposal, ASM Fellowship Proposal Included in this work is the proposal of work submitted to the American Society of Microbiology, for which I received a three-year fellowship. Some of the work was completed, though the majority of my dissertation and graduate work focused on the ALBA4 project discussed. Identification of Puf2-storage granule components that preserve Plasmodium sporozoite infectivity Background: Malaria causes approximately 627,000 deaths per year, and 3.4 billion people are at risk for contracting the parasite worldwide (WHO, 2013 World Malaria Report). The eukaryotic parasites responsible for this disease (genus Plasmodium) have been fundamentally characterized, with the genomes, transcriptomes, and proteomes of many species having been determined in the last decade (1). Many critical aspects of their life cycle and transmission have also been uncovered, and the molecular mechanisms that underlie them are rapidly being identified. Transmission of salivary gland sporozoites occurs when a female Anopheles mosquito takes a blood meal. This is a crucial bottleneck; only a few sporozoites are successfully transferred from vector to host and complete their passage to the liver. The Plasmodium parasites that successfully infect host hepatocytes dedifferentiate and undergo a complex development process, which leads to parasite release into the vasculature and the infection of red blood cells. A small fraction of blood stage parasites differentiate into sexual forms, which are potentially taken up by an uninfected female mosquito during a blood meal to perpetuate the parasite life cycle. Characterizations of the bottleneck between the vector/host and the parasites’ complex developmental process have elucidated parasite vulnerabilities. For example, the discovery that salivary gland sporozoites significantly up-regulate certain mRNA transcripts has led to effective, novel vaccines (2,3,4). Interestingly, many of these mRNA transcripts are not translated until sporozoites reach the host liver and productively infect a hepatocyte (5). Concurrent with the up-regulation of specific mRNAs, several RNA-binding proteins are also upregulated and accumulate as granules in the parasite, one being the RNA-binding protein

89

Puf2. As the parasite completes invasion of a hepatocyte and begins to dedifferentiate, Puf2 protein levels decrease dramatically, suggesting that Puf2 plays an important role in the transmission of sporozoites and establishment of liver stage development. Further, puf2- parasites lose their infectivity with prolonged residence times in the mosquito salivary gland, likely due to an imbalance of RNA abundance levels normally regulated by Puf2 (Fig. 1). Puf2, a member of the Pumilio-FBF (PUF) family of proteins, has a RNA-binding domain (RBD), which binds to mRNAs sequence specifically. PUF family proteins are known to modulate translation by either inhibiting mRNA circularization, blocking translation, or by increasing deadenylation and degradation of the mRNAs (6). These different RNA fates are known to be produced through the association of PUF/RNA granules with specific effector proteins (7). We hypothesize that Plasmodium Puf2 acts in a similar manner: by binding specific mRNAs and promoting a protective or degradative outcome for different RNA molecules based upon the association of different effector proteins. However, the composition of the novel granules that associate with Puf2 has yet to be uncovered. We therefore propose to determine the contribution of Puf2’s functions toward storage granule composition and parasite infectivity, and to elucidate the components of these novel Puf2 storage granules that accumulate in the sporozoite form of the genetically tractable rodent-infectious parasite Plasmodium yoelii. These studies will allow us to understand an underlying mechanism of sporozoite infectivity and transmission, and may identify novel interactions that will permit the formation of novel drug treatments and vaccines. Specific Aim 1: To determine the role of Puf2 in parasite infectivity. Through the use of targeted site-directed mutagenesis, Puf2 will be modified to disrupt RNA binding, protein binding, or both. The effects of these variants upon parasite infectivity will be assessed in vivo by the infection of mice with transgenic parasites that express these variants. Specific Aim 2: To dissect the protein and RNA components of Puf2 storage granules. Puf2

90 variants (Aim 1) will be immunoprecipitated using a novel in vivo biotinylation method, and the isolated granules will be subjected to mass spectrometry and RNA-seq to identify the protein and RNA components, respectively. Reciprocal labeling and immunoprecipitation (IP) of the identified effector proteins will also be performed to confirm storage granule composition. Aim 1: Our preliminary data indicates that Puf2 is necessary to maintain sporozoite infectivity over time (Fig.1, 8). As seen in puf2- parasites, infectivity is negatively correlated with duration of salivary gland residence (8). Other analyses have shown that sporozoites dedifferentiate prematurely in the salivary gland and resemble liver stage parasites (9). Premature dedifferentiation is accompanied by increased translation of transcripts that are up-regulated during the salivary gland sporozoite stage, but which are not translated in WT parasites. These data suggest that Puf2 binds to transcripts in sporozoites and protects/silences them or triggers their degradation. To determine the roles that Puf2’s ability to bind to RNA and/or other proteins play in parasite infectivity and storage granule composition, we have generated transgenic parasites that express full-length Puf2 or just its RBD alone fused with GFP at the C-terminus. In addition, we are also generating Puf2 variants with alanine substitutions at several residues that were identified by structural threading (modeled on PDB: 3K64) to be likely to facilitate RNA and protein interactions (K). These residues will be substituted in either an interaction loop (IL) shown to mediate protein/protein interactions in other species, in PUF repeat domains of the RBD, or in both the IL and RBD. After confirmation of the creation and isolation of transgenic parasites through genotyping PCR, mice will be infected with these transgenic sporozoites and monitored carefully for differences in the timing of the initiation of blood stage infection. Delays in the time to blood stage patency will indicate defects in parasite infectivity (e.g. 1 day delay = 10-fold defect), thus pinpointing RNA- and protein-binding sites on Puf2 that are crucial for its function (11). Further, we will be able to determine the localization of Puf2 throughout the parasite life cycle by both live- and immunofluorescence confocal microscopy. We hypothesize that substitutions in the RBD will inhibit specific RNA binding, meaning functional storage granules will also not form, RNA abundances will not be regulated, and that the parasite will become non-infectious similar to the puf2- parasite. We hypothesize that

91 mutations in the IL will prevent specific protein interactions with Puf2, and will inhibit formation of functional storage granules, and cause premature dedifferentiation and decreased infectivity. The ability of Puf2 to bind RNA may still protect these transcripts, but the effector proteins may not be able to bind and create the storage granule, which may lead to premature translation. Finally, with substitutions in both the RBD and IL, we expect Puf2 will not be able to bind RNAs or proteins specifically, leaving transcripts available for translation and leading to decreased infectivity as observed in puf2- parasites. However, if our Puf2 variants resemble WT parasites and remain infectious and form intact storage granules, the sites we have identified may not be critical to Puf2’s function. In this case, we will reevaluate the regions of conserved homology and test new variants. Aim 2: The possible players in Puf2 storage granules are currently unknown (Fig. 2); previously expected players, such as eIF2α and P-body proteins have been shown to form distinct granule bodies that do not co-localize with Puf2 (8). Therefore, a direct approach to identify proteins in storage granules will be used. In order to facilitate immunoprecipitation (IP) of Puf2 storage granules, a novel isolation strategy will be used, whereby transgenic parasites will be fused with GFP, a biotinylation enzyme (BirA), and a polymerized biotin accepting domain (3xAviTag) fused to the C-terminus of Puf2. Transgenic parasites expressing this fusion protein (GBA) will cause Puf2 to be biotinylated in situ, and thus allow capture of Puf2 granules with commercially available streptavidin-conjugated Dynabeads. Our laboratory has successfully constructed the GBA-tag, and shown that in vivo biotinylation can be detected with streptavidin-specific antibodies by immunofluorescence microscopy. This also allows for comparative IPs between wild-type Puf2 and its tagged variants (described in Aim 1), and background contaminants will be identified by independent expression of the GBA protein alone. If these complexes cannot be effectively captured through biotin/streptavidin interactions, antibodies against GFP will alternatively be used for these IPs, although we expect to see more background contamination

92 through this approach. This approach is feasible with our current GFP-fused Puf2 variants independent of the success of Aim 1. After IP, liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) will be performed on samples and GBA-only controls using a Thermo LTQ Orbitrap Velos ETD, which will allow peptides to be identified and protein identities to then be inferred. Presence of these proteins will also be assessed by western blotting with available and custom antisera. Similarly, RNA-seq will be performed on immunoprecipitated samples and GBA-only controls using an Illumina HiSeq 2500. This will allow identification of the proteins and RNAs directly involved with storage granules in WT parasites. Comparisons will again be made between WT Puf2 and Puf2 variants to pinpoint important Puf2 residues for granule composition and RNA binding. In order to rule out background contamination of samples, the LC-MS-MS results will be carefully compared to the contaminant repository for affinity purification- mass spectrometry data (termed the “CRAPome”) and our previous sporozoite proteomes (5,12). We expect the variants to act differently based on their substitutions; with the RBD substitution variant we expect that RNAs and RNA-dependent binding proteins will not be found in the storage granule and that these parasites will functionally resemble puf2- parasites. In the IL substitution variant, we expect to see some proteins no longer associating with Puf2, but anticipate that RNAs will remain in the complex. If these protein-protein interactions are important for translational repression, this parasite will also functionally resemble the puf2- parasite. Finally, in the RBD+IL substitution variant, we expect to observe the combined, and most severe, phenotype. This technique will also allow characterization of the storage granules as protective or degradative – or both. It will show us residues of the Puf2 RBD that are important for translational silencing and/or degradation, and the identification of proteins with known effector functions can be reciprocally tested to discern if two distinct granule populations (e.g. protective, degradative) exist. The work proposed above in both Aims will be carried out in parallel over the course of this fellowship. During Year One I will generate the Puf2 variant constructs and confirm transgenic populations. I will also optimize our IP protocols, and confirm them by western blotting. In Year Two I will perform the IP/MS and IP/RNA-seq experiments using Puf2-GBA.

93

During Year Three I will perform reciprocal IP experiments of identified proteins to discern between protective and degradative complexes. Importantly, this work allows for large-scale, practical biochemical characterization of Plasmodium sporozoites. Until recently, only IP of radiolabeled proteins has been possible. However, by using the GBA-fused protein, we anticipate that IP of non-radiolabeled proteins will now be accessible. This is supported by our recent advances in sporozoite purification, the validation of the GBA-tag, the reliable MS- detection of chemically biotinylated low-abundance proteins, as well as our ability to produce high quality total RNA-seq datasets from very low starting masses of RNA (5,8,13). These initial biochemical analyses on sporozoites will benefit the public by revealing mechanisms of infectivity and more of their basic biology. This work will further elucidate the biology of transmission events between vector/host, and allow us to perturb these events. Blocking transmission of parasites will greatly reduce the incidence of malaria and help eradicate the disease from those who live in or travel to endemic areas.

References 1. Gardner, MJ; Hall, Neil; Fung, E; White, O; Berriman, M: Hyman, RW; et al. 2002. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419:498-511. 2. Matuschewski, K; Ross, J; Brown, SM; Kaiser, K; Nussenzweig, V; Kappe, SHI. 2005. Infectivity-associated Changes in the Transcriptional Repertoire of the Malaria Parasite Sporozoite Stage. J Biol Chem 277:41948-41953. 3. Mikolajczak, SA; Silva-Rivera, H; Peng, X; Tarun, AS; Camargo, N; Jacobs-Lorena, V; et al. 2008. Distinct Malaria Parasite Sporozoites Reveal Transcriptional Changes That Cause Differential Tissue Infection Competence in the Mosquito Vector and mammalian Host. Mol Cell Biol 28(20):6196. 4. Finney, OC; Keitany, GJ; Smithers, H; Kaushansky, A; Kappe, S; Wang, R. 2014. Immunization with genetically attenuated P. falciparum parasites induces long-lived antibodies that efficiently block hepatocyte invasion by sporozoites. Vaccine 32(19):2135-8.

94

5. Lindner, SE; Swearingen, KE; Harupa, A; Vaughan, AM; Sinnis, P; Moritz, RL; Kappe, SHI. 2013. Total and Putative Surface Proteomics of Malaria Parasite Salivary sGland Sporozoites. Mol and Cell Proteomics 12:1127-1143. 6. Zamore, PD; Williamson, JR; Lehmann, R. 1997. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3:1421-1433. 7. Campbell, ZT; Menichelli, E; Friend, K; Wu, J; Kimble, J; Williamson, JR; Wickens, M. 2012. Identification of a conserved interface between PUF and CPEB proteins. J Biol Chem 287(22):18854-62. 8. Lindner, SE; Mikolajczak, SA; Vaughan, AM; Moon, W; Joyce, BR; Sullivan Jr, W; Kappe, SHI. 2013. Perturbations of Plasmodium Puf2 expression and RNA-seq of Puf2-deficient sporozoites reveal a critical role in maintatining RNA homeostasis and parasite transmissibility. Cell Microbiol 15(7)156-1283. 9. Gomes-Santos, CSS; Braks, J; Prudencio, M; Carret, C; Gomes, AR; Pain, A; et al. 2011. Transition of Plasmodium Sporozoites into Liver Stage-Like Forms Is Regulated by the RNA Binding Protein Pumilio. PLoS Pathog 7(5):e1002046. 10. Wu, J; Campbell, ZT; Menichelli, E; Wickens, M; Williamson JR. 2013. A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex J Mol Biol 425(4): 738-54. 11. Gantt, SM; Myung, JM; Briones, MR; Li, WD; Corey, EJ; Omura, S; Nussenzweig, V; Sinnis, P. 1998. Proteasome inhibitors block development of Plasmodium spp. Antimicrob Agents Chemother 42(10):2731-8. 12. Mellacheruvu, D; Wright, Z; Couzens, AL; Lambert, JP; St-Denis, NA; Li, T; et al. 2013. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10(8):730-736. 13. Kennedy, M; Fishbaugher, ME; Vaughan, AM; Patrapuvich, R; Boonhok, R; Yimamnuaychok, N; et al. 2012. A rapid and scalable density gradient purification method for Plasmodium sporozoites. Malar J 11:421.

95

APPENDIX E Contribution to “Perk Gene Dosage Regulates Glucose Homeostasis by Modulating Pancreatic β-Cell Functions” From January 2012 - September 2013, I worked in the laboratory of Dr. Douglas Cavener, under Dr. Rong Wang, at the time a fellow graduate student. During this time I contributed to the paper mentioned above, through experimentation, writing, and intelligent discussion. I was granted second authorship. My major contribution was to Figure 4 of this paper, by carrying out the BrdU experiment mentioned below. This was to determine if there was a significant expansion of β-cells in the pancreas in Perk heterozygotes. I found that there was an increase in β-cell proliferation in these mice compared to their WT littermates. Citation numbers are consistent with this dissertation. Below is Figure 4:

“β-cell number is increased in Perk heterozygotes due to elevated β-cell proliferation”

Unlike P17 mice, Perk+/− mice at P50 did not show increased expression of insulin mRNA level (Fig 4B) or protein level per β-cell (Fig 3C). However, P50 Perk+/− mice had substantially higher β-cell number (Fig 3D). To confirm this observation, β-cell number was estimated using the expression of mRNA of two genes, insulin II and Glut2, after previously published methods [161, 162]. Since both genes are exclusively expressed in β-cells, their mRNA levels in whole pancreata are directly proportional to β-cell number [206, 2017]. Perk+/− mice at P50 had higher total insulin (P<0.05, Fig 4A) and total Glut2 (p = 0.08) mRNA in the total pancreas compared to wild-type mice whereas expression levels of these two genes in islets were not different between genotypes (Fig 4A), reflecting a 56%–69% increase in total β-cells in Perk+/− (Fig 4A) with equivalent level of expression of insulin II and Glut2 per β-cell.

96

P50 Perk+/− mice exhibit higher β-cell number due to elevated β-cell proliferation.

To further investigate the reason for increased β-cell number in P50 Perk+/− mice, β-cell proliferation was determined by BrdU incorporation. β-cell proliferation was found to be significantly increased in P50 Perk+/− mice compared to WT controls (Fig 4B). We also examined

β-cell proliferation at four other developmental time points and found that Perk+/− exhibited elevated proliferation at P30 and P50 but not earlier or later time points (Fig 4B), indicating that enhanced proliferation was transient and corresponded to the time period when β-cell number was increased in Perk+/− mice. In addition, β-cell death was estimated using TUNEL assay and found to be negligible and not different between Perk genotypes (data not shown).”

97

BIBLIOGRAPHY 1. Correll C, Bartek J, Dundr M. The Nucleolus: a multiphase condensate balancing ribosome synthesis and translational capacity in health, aging, and ribosomopathies. Cell. 2019;8:869-887. 2. Feric M, Vaidya N, Harmon TS, Mitrea DM, Zhu L, Richardson TM, Kriwacki RW, Pappu RV, Brangwynne CP. Coexisting Liquid Phases Underlie Nucleolar Subcompartments. Cell. 2016;165:1686-1697. 3. Denissov S, Lessard F, Mayer C, Stefanovsky V, van Driel M, Grummt I, Moss T, Stunnenberg HG. A model for the topology of active ribosomal RNA genes. EMBO Rep. 2011;12:231-237. 4. Dundr, M. Nuclear bodies: Multifunctional companions of the genome. Curr Opin Cell Biol. 2012;24:415-422. 5. Koberna K, Malinsky J, Pliss A, Masata M, Vecerova J, Fialova M, Bednar J, Raska I. Ribosomal genes in focus: New transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ. J Cell Biol. 2002;157:743-748. 6. Schofer C, Weipoltshammer K. Nucleolus and chromatin. Histochem Cell Biol. 2018;150:209-225. 7. Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, et al. Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol. 2018;28:420-435. 8. Gomes E, Shorter J. The molecular language of membraneless organelles. J Biol Chem. 2019;294:7115-7127. 9. Sawyer IA, Bartek J, Dundr M. Phase separated microenvironments inside the cell nucleus are linked to disease and regulate epigenetic state, transcription and RNA processing. Semin Cell Dev Biol 2019;90:94-103. 10. Wang Z, Zhang H. Phase Separation, Transition, and Autophagic Degradation of Proteins in Development and Pathogenesis. Trends Cell Biol 2019;29:417-427. 11. Woodru JB, Hyman AA, Boke E. Organization and Function of Non-dynamic Biomolecular Condensates. Trends Biochem Sci. 2018;43:81-94.

98

12. Michieletto D, Gilbert N. Role of nuclear RNA in regulating chromatin structure and transcription. Curr Opin Cell Biol. 2019;58:120-125. 13. Turoverov KK, Kuznetsova IM, Fonin AV, Darling AL, Zaslavsky BY, Uversky VN. Stochasticity of Biological Soft Matter: Emerging Concepts in Intrinsically Disordered Proteins and Biological Phase Separation. Trends Biochem Sci. 2019;44:716-728. 14. Uversky VN. Intrinsically disordered proteins in overcrowded milieu: Membrane-less organelles, phase separation, and intrinsic disorder. Curr Opin Struct Biol. 2017; 44:18- 30. 15. Chujo T, Hirose T. Nuclear Bodies Built on Architectural Long Noncoding RNAs: Unifying Principles of Their Construction and Function. Mol Cells. 2017;40:889-896. 16. Chujo T, Yamazaki T, Kawaguchi T, Kurosaka S, Takumi T, Nakagawa S, Hirose T. Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs. EMBO J. 2017;36:1447-1462. 17. Wei MT, Elbaum-Garfinkle S, Holehouse AS, Chen CC, Feric M, Arnold CB, Priestley RD, Pappu RV, Brangwynne CP. Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles. Nat Chem. 2017;9:1118-1125. 18. O’Neil D, Glowatz H, Schlumpberger M. Ribosomal RNA depletion for efficient use of RNA-seq capacity. Curr Protoc Mol Biol. 2013;103. 19. Pelletier J, Thomas G, Volarevic S. Ribosome biogenesis in cancer: New players and therapeutic avenues. Nat Rev Cancer. 2018;18:51-63. 20. Bassler J, Hurt E. Eukaryotic Ribosome Assembly. Annu Rev Biochem. 2019;88:281-306. 21. Klinge S, Woolford JL Jr. Ribosome assembly coming into focus. Nat Rev Mol Cell Biol. 2019;20:116-131. 22. Frottin F, Schueder F, Tiwary S, Gupta R, Korner R, Schlichthaerle T, Cox J, Jungmann R, Hartl FU, Hipp MS. The nucleolus functions as a phase-separated protein quality control compartment. Science. 2019;365:342-347. 23. Ghalei H, Trepreau J, Collins JC, Bhaskaran H, Strunk BS, Karbstein K. The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation. Mol Cell. 2017;67:990-1000.e3.

99

24. Lebaron S, Schneider C, van Nues RW, Swiatkowska A, Walsh D, Bottcher B, Granneman S, Watkins NJ, Tollervey D. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nat Struct Mol Biol. 2012;19:744-753. 25. Strunk BS, Novak MN, Young CL, Karbstein K. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits. Cell. 2012;150:111-121. 26. Sawyer IA, Dundr M. Nuclear bodies: Built to boost. J Cell Biol. 2016;213:509-511. 27. Stroberg W, Schnell S. Do Cellular Condensates Accelerate Biochemical Reactions? Lessons from Microdroplet Chemistry. Biophys J. 2018;115:3-8. 28. Brangwynne CP, Mitchison TJ, Hyman AA. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc Natl Acad Sci USA. 2011;108:4334-4339. 29. Mitrea DM, Cika JA, Guy CS, Ban D, Banerjee PR, Stanley CB, Nourse A, Deniz AA, Kriwacki RW. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. eLife. 2016;5:e13571. 30. Fay MM, Anderson PJ. The Role of RNA in Biological Phase Separations. J Mol Biol. 2018;430:4685-4701. 31. Boisvert FM, van Koningsbruggen S, Navascues J, Lamond AI. The multifunctional nucleolus. Nat Rev Mol Cell Biol. 2007;8(7):574-585. 32. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M. Nucleolar proteome dynamics. Nature. 2005;433(7021):77-83. 33. Desnoyers S, Kaufmann SH, and Poirier GG. Alteration of the nucleolar localization of poly(ADP-ribose) polymerase upon treatment with transcription inhibitors. Exp Cell Res. 1996;227(1): 146-153. 34. Thielmann HW, Popanda O, Staab HJ. Subnuclear distribution of DNA topoisomerase I and Bax protein in normal and xeroderma pigmentosum fibroblasts after irradiation with UV light and gamma rays or treatment with topotecan. J Cancer Res Clin Oncol. 1999;125(3-4):193-208.

100

35. Chan PK, Bloom DA, and Hoang TT. The N-terminal half of NPM dissociates from nucleoli of HeLa cells after anticancer drug treatments. Biochem Biophys Res Commun. 1999;264(1): 305-309. 36. Matthews DA. Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm. J Virol. 2001;75(2):1031-1038. 37. Kim JY, Seok KO, Kim YJ, Bae WK, Lee S, Park JH. Involvement of GLTSCR2 in the DNA Damage Response. Am J Pathol. 2011;179(3):1257-1264. 38. Yang K, Yang J, Yi J. Nucleolar Stress: hallmarks, sensing mechanism and diseases. Cell Stress. 2018;2(6):125-140. doi: 10.15698/cst2018.06.139. 39. Ogbadoyi E, Ersfeld K, Robinson D, Sherwin T, Gull K. Architecture of the Trypanosoma brucei nucleus during interphase and mitosis. Chromosoma. 2000;108:501-513. 40. Lopez-Velazquez G, Hernandez R, Lopez-Villasenor I, Reyes-Vivas H, Segura-Valdez ML, Jimenez-Garcia LF. Electron microscopy analysis of the nucleolus of Trypanosoma cruzi. Microsc Microanal. 2005;11:293-299. 41. Nepomuceno-Mejia T, Lara-Martinez R, Cevallos AM, Lopez-Villasenor I, Jimenez-Garcia LF, Hernandez R. The Trypanosoma cruzi nucleolus: A morphometrical analysis of cultured epimastigotes in the exponential and stationary phases. FEMS Microbiol Lett. 2010;313:41-46. 42. Thiry M, Lafontaine DL. Birth of a nucleolus: The evolution of nucleolar compartments. Trends Cell Biol. 2005;15:194-199. 43. Kedersha N, Anderson P. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. Biochem Soc Trans. 2002;30(Pt 6):963-9. Epub 2002/11/21. doi: 10.1042. 44. Gilks N, Kedersha N, Ayodele M, Shen L, Stoecklin G, Dember LM, Anderson P. Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol Biol Cell. 2004;15:5383–5398. 45. Buchan JR, Parker R. Eukaryotic stress granules: the ins and outs of translation. Molecular cell. 2009;36(6):932-41. Epub 2010/01/13. doi:10.1016/j.molcel.2009.11.020.

101

46. Kimball SR, Horetsky RL, Ron D, Jefferson LS, Harding HP. Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. American journal of physiology Cell physiology. 2003;284(2):C273-84. Epub 2002/10/22. doi:10.1152/ajpcell.00314.2002. 47. Jain S, Wheeler Joshua R, Walters Robert W, Agrawal A, Barsic A, Parker R. ATPase- Modulated Stress Granules Contain a Diverse Proteome and Substructure. Cell. 2016;164:1-12. doi: 10.1016/j.cell.2015.12.038. 48. Jain S, Parker R. The discovery and analysis of P Bodies. Advances in experimental medicine and biology. 2013;768:23-43. Epub 2012/12/12. doi: 10.1007/978-1-4614- 5107-5_3. 49. Decker CJ, Parker R. P-bodies and stress granules: possible roles in the control of translation and mRNA degradation. Cold Spring Harbor perspectives in biology. 2012;4(9):a012286. Epub 2012/07/06. doi: 10.1101/cshperspect.a012286. 50. Hoyle NP, Castelli LM, Campbell SG, Holmes LE, Ashe MP. Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies. The Journal of cell biology. 2007;179(1):65-74. Epub 2007/10/03. doi: 10.1083/jcb.200707010. 51. Kedersha N, Stoecklin G, Ayodele M, Yacono P, Lykke-Andersen J, Fritzler MJ, et al. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. The Journal of cell biology. 2005;169(6):871-84. Epub 2005/06/22. doi:10.1083/jcb.200502088. 52. Teixeira D, Sheth U, Valencia-Sanchez MA, Brengues M, Parker R. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA. 2005;11(4):371-82. Epub 2005/02/11. doi: 10.1261/rna.7258505. 53. Protter DSW, Parker R. Principles and Properties of Stress Granules. Trends Cell Biol. 2016 Sep;26(9):668-79. doi: 10.1016/j.tcb.2016.05.004. Epub 2016 Jun 9. 54. Tourriere H, et al. TheRasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 2003;160, 823–831.

102

55. Kedersha N, et al. G3BP–Caprin1–USP10 complexes mediate stress granule condensation and associate with 40S subunits. J. Cell Biol. 2016;212, 845–860. 56. Gilks, N. et al. Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol. Biol. Cell 2004;15, 5383–5398. 57. Nott, T.J.et al. Phase transition of a disordered nuage protein generates environmentally responsive membrane-less organelles. Mol. Cell 2015;57, 936-947. 58. Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl. Acad. Sci. USA. 2015;112, 7189-7194. 59. Patel, A. et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 2015;162, 1066–1077. 60. Lin, Y. et al. (2015) Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell 60, 208-219. 61. Molliex, A. et al. (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123-133. 62. Tsai, N.P. et al. Regulation of stress granule dynamics by Grb7 and FAK signalling pathway. EMBO J. 2008;27, 715–726. 63. Goulet, I. et al. TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules. Hum. Mol. Genet. 2008;17, 3055-3074. 64. Kwon, S. et al The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 2007;21, 3381-3394. 65. Hilliker A, et al. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F–mRNA complex. Mol Cell, 2011;43:962-972. 66. Buchan JR, et al. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell. 2013;153:1461–1474. 67. Meyer H, Weihl CC. The VCP/p97 system at a glance: connecting cellular function to disease pathogenesis. J Cell Sci. 2014;127:3877–3883.

103

68. Adeli K. Translational control mechanisms in metabolic regulation: critical role of RNA binding proteins, microRNAs, and cytoplasmic RNA granules. Am J Physiol Endocrinol Metab. 2011;301:E1051–1064. 69. Thomas MG, Loschi M, Desbats MA, Boccaccio GL. RNA granules: the good, the bad and the ugly. Cell Signal. 2011;23:324–334. 70. Dewey CM, Cenik B, Sephton CF, Dries DR, Mayer P, 3rd, Good SK, Johnson BA, Herz J, Yu G. TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor. Mol Cell Biol. 2011;31:1098–1108. 71. Anderson P, Kedersha N. Stress granules: the Tao of RNA triage. Trends Biochem Sci. 2008;33:141–150. 72. Waelter S, Boeddrich A, Lurz R, Scherzinger E, Lueder G, Lehrach H, Wanker EE. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol Biol Cell. 2001;12:1393–1407. 73. Goggin K, Beaudoin S, Grenier C, Brown AA, Roucou X. Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response. Biochim Biophys Acta. 2008;1783:479–491. 74. Liu-Yesucevitz L, Bilgutay A, Zhang YJ, Vanderweyde T, Citro A, Mehta T, Zaarur N, McKee A, Bowser R, Sherman M, Petrucelli L, Wolozin B. Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One. 2010;5:e13250. 75. Kampers T, Friedhoff P, Biernat J, Mandelkow EM, Mandelkow E. RNA stimulates aggregation of microtubule-associated protein tau into Alzheimer-like paired helical filaments. FEBS Lett. 1996;399:344–349. 76. Wolozin B. Regulated protein aggregation: stress granules and neurodegeneration. Mol Neurodegener. 2012;7:56. 77. Liu-Yesucevitz L, Bassell GJ, Gitler AD, Hart AC, Klann E, Richter JD, Warren ST, Wolozin B. Local RNA translation at the synapse and in disease. J Neurosci. 2011;31:16086– 16093.

104

78. Liu-Yesucevitz L, Bilgutay A, Zhang YJ, Vanderweyde T, Citro A, Mehta T, Zaarur N, McKee A, Bowser R, Sherman M, Petrucelli L, Wolozin B. Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue. PLoS One. 2010;5:e13250. 79. Vanderweyde T, Youmans K, Liu-Yesucevitz L, Wolozin B. 2013. The roles stress granule and RNA binding proteins in neurodegeneration. Gerontology. 2013; 59(6):10.1159/000354170. 80. McDonald KK, Aulas A, Destroismaisons L, Pickles S, Beleac E, Camu W, Rouleau GA, Vande Velde C. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1. Hum Mol Genet. 2011;20:1400–1410. 81. Nussbaum JM, Seward ME, Bloom GS. Alzheimer disease: a tale of two prions. Prion. 2013;7(1): 14–9. doi:10.4161/pri.22118. 82. Pulawski W, Ghoshdastider U, Andrisano V, Filipek S. Ubiquitous amyloids. Applied Biochemistry and Biotechnology. 2012;166(7): 1626–43. 83. Vanderweyde T, Yu H, Varnum M, Liu-Yesucevitz L, Citro A, Ikezu T, Duff K, Wolozin B. Contrasting Pathology of Stress Granule Proteins TIA-1 and G3BP in Tauopathies. J Neurosci. 2012;32:8270–8283. 84. Hoover BR, Reed MN, Su J, Penrod RD, Kotilinek LA, Grant MK, Pitstick R, Carlson GA, Lanier LM, Yuan LL, Ashe KH, Liao D. Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron. 2010;68:1067– 1081. 85. Walters RW, et al. Differential effects of Ydj1 and Sis1 on Hsp70-mediated clearance of stress granules in Saccharomyces cerevisiae. RNA. 2015;21:1660-1671. 86. Cipolat MS, et al. Autophagy in motor neuron disease: key pathogenetic mechanisms and therapeutic targets. Mol Cell Neurosci. 2016;72:84-90. 87. Li S, et al. Genetic interaction of hnRNPA2B1 and DNAJB6 in a Drosophila model of multisystem proteinopathy. Hum Mol Genet. 2016;25:936-950. 88. Lin Y, et al. Formation and maturation of phase-separated liquid droplets by RNA- binding proteins. Mol Cell. 2015;60:208-219.

105

89. Patel A, et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 2015;162:1066-1077. 90. Ramaswami M, et al. Altered ribostasis: RNA–protein granules in degenerative disorders. Cell. 2013;154:727-736. 91. Freibaum BD, et al. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015;525:129-133. 92. Ling SC, et al. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron. 2013;79:416-438. 93. Zhang K, et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015;525:56-61. 94. (WHO) WHO. World Malaria Report 2019. 2019. 95. Dantzler KW, Ravel DB, Brancucci NM, Marti M. Ensuring transmission through dynamic host environments: host-pathogen interactions in Plasmodium sexual development. Current opinion in microbiology. 2015;26:17-23. Epub 2015/04/14. doi: 10.1016/j.mib.2015.03.005. 96. Wu Y, Sinden RE, Churcher TS, Tsuboi T, Yusibov V. Development of malaria transmission-blocking vaccines: from concept to product. Advances in parasitology. 2015;89:109-52. 97. Khan SM, Janse CJ, Kappe SH, Mikolajczak SA. Genetic engineering of attenuated malaria parasites for vaccination. Current opinion in biotechnology. 2012;23(6):908-16. Epub 2012/05/09. doi: 10.1016/j.copbio.2012.04.003. 98. Muller K, Matuschewski K, Silvie O. The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite. PLoS One. 2011;6(5):e19860. Epub 2011/06/16. doi: 10.1371/journal.pone.0019860. 99. Bunnik EM, Chung DW, Hamilton M, Ponts N, Saraf A, Prudhomme J, et al. Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum. Genome biology. 2013;14(11):R128. Epub 2013/11/26. doi: 10.1186/gb-2013-14-11-r128.

106

100. Oakley MS, Kumar S, Anantharaman V, Zheng H, Mahajan B, Haynes JD, Moch JK, Fairhurst R, McCutchan TF, Aravind L. Molecular factors and biochemical pathways induced by febrile temperature in intraerythrocytic Plasmodium falciparum parasites. Infect Immun. 2007 Apr;75(4):2012-25. Epub 2007 Feb 5. 101. Oakley MS, Gerald N, Anantharaman V, Gao Y, Majam V, Mahajan B, Pham PT, Lotspeich-Cole L, Myers TG, McCutchan TF, Morris SL, Aravind L, Kumar S. Radiation- induced cellular and molecular alterations in asexual intraerythrocytic Plasmodium falciparum. J Infect Dis. 2013 Jan 1;207(1):164-74. doi: 10.1093/infdis/jis645. Epub 2012 Oct 24. 102. Swisher KD, Parker R. Localization to, and effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on stress granules in Saccharomyces cerevisiae. PLoS One. 2010 Apr 2;5(4):e10006. doi: 10.1371/journal.pone.0010006. 103. Hall N, Karras M, Raine JD, Carlton JM, Kooij TW, Berriman M, et al. A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005;307(5706):82-6. Epub 2005/01/08. doi: 10.1126/science.1103717. 104. Paton MG, Barker GC, Matsuoka H, Ramesar J, Janse CJ, Waters AP, et al. Structure and expression of a post-transcriptionally regulated malaria gene encoding a surface protein from the sexual stages of Plasmodium berghei. Molecular and biochemical parasitology. 1993;59(2):263-75. Epub 1993/06/01. 105. Yuda M, Iwanaga S, Kaneko I, Kato T. Global transcriptional repression: An initial and essential step for Plasmodium sexual development. Proceedings of the National Academy of Sciences of the United States of America. 2015;112(41):12824-9. Epub 2015/09/30. doi: 10.1073/pnas.1504389112. 106. Lindner SE, Mikolajczak SA, Vaughan AM, Moon W, Joyce BR, Sullivan WJ, et al. Perturbations of Plasmodium Puf2 expression and RNA-seq of Puf2-deficient sporozoites reveal a critical role in maintaining RNA homeostasis and parasite transmissibility. Cellular Microbiology. 2013;15(7):1266-83. doi: 10.1111/cmi.12116.

107

107. Mair GR, Braks JA, Garver LS, Wiegant JC, Hall N, Dirks RW, et al. Regulation of sexual development of Plasmodium by translational repression. Science. 2006;313(5787):667- 9. Epub 2006/08/05. doi: 10.1126/science.1125129. 108. Lindner SE, Swearingen KE, Harupa A, Vaughan AM, Sinnis P, Moritz RL, et al. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Molecular & cellular proteomics : MCP. 2013;12(5):1127-43. Epub 2013/01/18. doi: 10.1074/mcp.M112.024505. 109. Vermeulen AN, Ponnudurai T, Beckers PJ, Verhave JP, Smits MA, Meuwissen JH. Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito. The Journal of experimental medicine. 1985;162(5):1460-76. Epub 1985/11/01. 110. Siden-Kiamos I, Vlachou D, Margos G, Beetsma A, Waters AP, Sinden RE, et al. Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei. Journal of cell science. 2000;113 Pt 19:3419-26. Epub 2000/09/14. 111. Mueller AK, Labaied M, Kappe SH, Matuschewski K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature. 2005;433(7022):164-7. Epub 2004/12/08. doi: 10.1038/nature03188. 112. Mueller AK, Camargo N, Kaiser K, Andorfer C, Frevert U, Matuschewski K, et al. Plasmodium liver stage developmental arrest by depletion of a protein at the parasite- host interface. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(8):3022-7. Epub 2005/02/09. doi: 10.1073/pnas.0408442102. 113. Guerreiro A, Deligianni E, Santos JM, Silva PA, Louis C, Pain A, et al. Genome-wide RIP- Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte. Genome biology. 2014;15(11):493. doi: 10.1186/s13059-014-0493-0. 114. Vembar SS, Macpherson CR, Sismeiro O, Coppee JY, Scherf A. The PfAlba1 RNA-binding protein is an important regulator of translational timing in Plasmodium falciparum blood stages. Genome biology. 2015;16(1):212. Epub 2015/09/30. doi: 10.1186/s13059-015- 0771-5.

108

115. Gomes-Santos CSS, Braks J, Prudencio M, Carret C, Gomes AR, Pain A, et al. Transition of Plasmodium Sporozoites into Liver Stage-Like Forms Is Regulated by the RNA Binding Protein Pumilio. PLoS Pathog. 2011;7(5):e1002046. doi: 10.1371/journal.ppat.1002046. 116. Mair GR, Lasonder E, Garver LS, Franke-Fayard BM, Carret CK, Wiegant JC, et al. Universal features of post-transcriptional gene regulation are critical for Plasmodium zygote development. PLoS Pathog. 2010;6(2):e1000767. Epub 2010/02/20. doi: 10.1371/journal.ppat.1000767. 117. Chene A, Vembar SS, Riviere L, Lopez-Rubio JJ, Claes A, Siegel TN, et al. PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites. Nucleic acids research. 2012;40(7):3066-77. Epub 2011/12/15. doi: 10.1093/nar/gkr1215. 118. Goyal M, Alam A, Iqbal MS, Dey S, Bindu S, Pal C, et al. Identification and molecular characterization of an Alba-family protein from human malaria parasite Plasmodium falciparum. Nucleic acids research. 2012;40(3):1174-90. Epub 2011/10/19. doi: 10.1093/nar/gkr821. 119. BA Zhang M, Joyce BR, Sullivan WJ, Jr., Nussenzweig V. Translational control in Plasmodium and toxoplasma parasites. Eukaryotic cell. 2013;12(2):161-7. Epub 2012/12/18. doi: 10.1128/ec.00296-12. 120. Cui L, Lindner S, Miao J. Translational regulation during stage transitions in malaria parasites. Annals of the New York Academy of Sciences. 2015;1342:1-9. Epub 2014/11/13. doi: 10.1111/nyas.12573. 121. Wickens M, Bernstein DS, Kimble J, Parker R. A PUF family portrait: 3'UTR regulation as a way of life. Trends Genet. 2002 Mar;18(3):150-7. 122. Murata Y, Wharton RP. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell. 1995 Mar 10;80(5):747-56. 123. Zhang B, Gallegos M, Puoti A, Durkin E, Fields S, Kimble J, Wickens MP. A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature. 1997 Dec 4;390(6659):477-84.

109

124. Luu VD, Brems S, Hoheisel JD, Burchmore R, Guilbride DL, Clayton C. Functional analysis of Trypanosoma brucei PUF1. Mol Biochem Parasitol. 2006 Dec;150(2):340-9. Epub 2006 Oct 18. 125. Folgueira C, Martinez-Bonet M, Requena JM.The Leishmania infantum PUF proteins are targets of the humoral response during visceral leishmaniasis. BMC Res Notes. 2010 Jan 21;3:13. doi: 10.1186/1756-0500-3-13. 126. Liu M, Miao J, Liu T, Sullivan WJ Jr, Cui L1, Chen X. Parasit Vectors. Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii. 2014 Mar 31;7:141. doi: 10.1186/1756-3305-7-141. 127. Miao J, Li J, Fan Q, Li X, Li X, Cui L. The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum. Journal of cell science. 2010;123(Pt 7):1039-49. Epub 2010/03/04. doi: 10.1242/jcs.059824. 128. Miao J, Fan Q, Parker D, Li X, Li J, Cui L. Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites. PLoS Pathog. 2013;9(4):e1003268. Epub 2013/05/03. doi: 10.1371/journal.ppat.1003268. 129. Kramer S, Marnef A, Standart N, Carrington M. Inhibition of mRNA maturation in Trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate. Journal of cell science. 2012;125(Pt 12):2896-909. Epub 2012/03/01. doi: 10.1242/jcs.099275. 130. Dupe A, Dumas C, Papadopoulou B. An Alba-domain protein contributes to the stage- regulated stability of amastin transcripts in Leishmania. Molecular microbiology. 2014;91(3):548-61. Epub 2014/03/14. doi: 10.1111/mmi.12478. 131. Mikolajczak SA, Silva-Rivera H, Peng X, Tarun AS, Camargo N, Jacobs-Lorena V, et al. Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host. Molecular and cellular biology. 2008;28(20):6196-207. doi: 10.1128/MCB.00553-08.

110

132. Reddy BP, Shrestha S, Hart KJ, Liang X, Kemirembe K, Cui L, et al. A bioinformatic survey of RNA-binding proteins in Plasmodium. BMC genomics. 2015;16(1):890. Epub 2015/11/04. doi: 10.1186/s12864-015-2092-1. 133. Guerrier-Takada C, Eder PS, Gopalan V, Altman S. Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P. RNA. 2002;8:290-295. 134. Keeling PJ, Rayner JC. The origins of malaria: there are more things in heaven and earth. Parasitology. 2015;142 Suppl 1:S16-25. Epub 2014/06/26. doi: 10.1017/s0031182014000766. 135. Barkan A, Klipcan L, Ostersetzer O, Kawamura T, Asakura Y, Watkins KP. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein. Rna. 2007;13(1):55-64. Epub 2006/11/16. doi: 10.1261/rna.139607. 136. Laurens N, Driessen RP, Heller I, Vorselen D, Noom MC, Hol FJ, et al. Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA. Nat Commun. 2012;3:1328. Epub 2012/12/29. doi: 10.1038/ncomms2330. 137. Jelinska C, Petrovic-Stojanovska B, Ingledew WJ, White MF. Dimer-dimer stacking interactions are important for nucleic acid binding by the archaeal chromatin protein Alba. The Biochemical journal. 2010;427(1):49-55. Epub 2010/01/20. doi: 10.1042/bj20091841. 138. Mons B. Intra erythrocytic differentiation of Plasmodium berghei. Acta Leidensia. 1986;54:1-124. Epub 1986/01/01. 139. Subota I, Rotureau B, Blisnick T, Ngwabyt S, Durand-Dubief M, Engstler M, et al. ALBA proteins are stage regulated during trypanosome development in the tsetse fly and participate in differentiation. Molecular biology of the cell. 2011;22(22):4205-19. Epub 2011/10/04. doi: 10.1091/mbc.E11-06-0511. 140. Mani J, Guttinger A, Schimanski B, Heller M, Acosta-Serrano A, Pescher P, et al. Alba- domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery. PLoS One. 2011;6(7):e22463. doi: 10.1371/journal.pone.0022463.

111

141. Matthews KR. The developmental cell biology of Trypanosoma brucei. J cell sci. 2005;118(Pt 2):283-290. doi: 10.1242/jcs.01649. 142. Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, et al. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002;419(6906):537-42. Epub 2002/10/10. doi: 10.1038/nature01111. 143. Kennedy M, Fishbaugher ME, Vaughan AM, Patrapuvich R, Boonhok R, Yimamnuaychok N, et al. A rapid and scalable density gradient purification method for Plasmodium sporozoites. Malaria journal. 2012;11:421. Epub 2012/12/19. doi: 10.1186/1475-2875- 11-421. 144. Swearingen KE, Lindner SE, Shi L, Shears MJ, Harupa A, Hopp CS, et al. Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics. PLoS Pathog. 2016;12(4):e1005606. Epub 2016/04/30. doi: 10.1371/journal.ppat.1005606. 145. Miller JL, Harupa A, Kappe SH, Mikolajczak SA. Plasmodium yoelii macrophage migration inhibitory factor is necessary for efficient liver-stage development. Infection and immunity. 2012;80(4):1399-407. doi: 10.1128/IAI.05861-11. 146. Deutsch EW, Mendoza L, Shteynberg D, Slagel J, Sun Z, Moritz RL. Trans-Proteomic Pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics. Proteomics Clinical applications. 2015;9(7-8):745-54. Epub 2015/01/30. doi: 10.1002/prca.201400164. 147. Kessner D, Chambers M, Burke R, Agus D, Mallick P. ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics. 2008;24(21):2534-6. Epub 2008/07/09. doi: 10.1093/bioinformatics/btn323. 148. Craig R, Beavis RC. TANDEM: matching proteins with tandem mass spectra. Bioinformatics. 2004;20(9):1466-7. Epub 2004/02/21. doi: 10.1093/bioinformatics/bth092.

112

149. Eng JK, Jahan TA, Hoopmann MR. Comet: an open-source MS/MS sequence database search tool. Proteomics. 2013;13(1):22-4. Epub 2012/11/14. doi: 10.1002/pmic.201200439. 150. Shteynberg D, Deutsch EW, Lam H, Eng JK, Sun Z, Tasman N, et al. iProphet: multilevel integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates. Molecular & cellular proteomics : MCP. 2011;10(12):M111.007690. Epub 2011/08/31. doi: 10.1074/mcp.M111.007690. 151. Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Analytical chemistry. 2003;75(17):4646-58. Epub 2003/11/25. 152. Choi H, Larsen B, Lin ZY, Breitkreutz A, Mellacheruvu D, Fermin D, et al. SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat Methods. 2011;8(1):70-3. Epub 2010/12/07. doi: 10.1038/nmeth.1541. 153. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome biology. 2013;14(4):R36. Epub 2013/04/27. doi: 10.1186/gb-2013-14-4-r36. 154. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature biotechnology. 2010;28(5):511-5. Epub 2010/05/04. doi: 10.1038/nbt.1621. 155. Pimentel J, Boccaccio GL. Translation and silencing in RNA granules: a tale of sand grains. Front Mol Neurosci. 2014;7:68. doi: 10.3389/fnmol.2014.00068. 156. Mikolajczak SA, Aly AS, Dumpit RF, Vaughan AM, Kappe SH. An efficient strategy for gene targeting and phenotypic assessment in the Plasmodium yoelii rodent malaria model. Molecular and biochemical parasitology. 2008;158(2):213-6. Epub 2008/02/05. doi: 10.1016/j.molbiopara.2007.12.006. 157. Lindner SE, Llinas M, Keck JL, Kappe SH. The primase domain of PfPrex is a proteolytically matured, essential enzyme of the apicoplast. Molecular and biochemical parasitology. 2011;180(2):69-75. doi: 10.1016/j.molbiopara.2011.08.002.

113

158. Malaria Methods and Protocols. Totowa, New Jersey: Humana Press; 2002. 159. Buchan JR, Muhlrad D, Parker R. P bodies promote stress granule assembly in Saccharomyces cerevisiae. The Journal of cell biology. 2008;183(3):441-55. Epub 2008/11/05. doi: 10.1083/jcb.200807043. 160. Sutherland JM, McLaughlin EA, Hime GR, Siddall NA. The Musashi family of RNA binding proteins: master regulators of multiple stem cell populations. Advances in experimental medicine and biology. 2013;786:233-45. Epub 2013/05/23. doi: 10.1007/978-94-007- 6621-1_13. 161. Mazumder B, Seshadri V, Fox PL. Translational control by the 3'-UTR: the ends specify the means. Trends Biochem Sci. 2003;28(2):91-8. Epub 2003/02/11. doi: 10.1016/s0968- 0004(03)00002-1. 162. Kedersha N, Cho MR, Li W, Yacono PW, Chen S, Gilks N, et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. The Journal of cell biology. 2000;151(6):1257-68. Epub 2000/12/21. 163. Huttelmaier S, Zenklusen D, Lederer M, Dictenberg J, Lorenz M, Meng X, et al. Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature. 2005;438(7067):512-5. Epub 2005/11/25. doi: 10.1038/nature04115. 164. Huang Y, Steitz JA. SRprises along a messenger's journey. Molecular cell. 2005;17(5):613-5. Epub 2005/03/08. doi: 10.1016/j.molcel.2005.02.020. 165. Was 155. Twyffels L, Gueydan C, Kruys V. Shuttling SR proteins: more than splicing factors. The FEBS journal. 2011;278(18):3246-55. Epub 2011/07/29. doi: 10.1111/j.1742- 4658.2011.08274.x. 166. WAS 156. Eshar S, Altenhofen L, Rabner A, Ross P, Fastman Y, Mandel-Gutfreund Y, et al. PfSR1 controls alternative splicing and steady-state RNA levels in Plasmodium falciparum through preferential recognition of specific RNA motifs. Molecular microbiology. 2015;96(6):1283-97. doi: 10.1111/mmi.13007. 167. Eshar S, Allemand E, Sebag A, Glaser F, Muchardt C, Mandel-Gutfreund Y, et al. A novel Plasmodium falciparum SR protein is an alternative splicing factor required for the

114

parasites' proliferation in human erythrocytes. Nucleic acids research. 2012;40(19):9903-16. Epub 2012/08/14. doi: 10.1093/nar/gks735. 168. Komaki-Yasuda K, Okuwaki M, Nagata K, Kawazu S, Kano S. Identification of a novel and unique transcription factor in the intraerythrocytic stage of Plasmodium falciparum. PLoS One. 2013;8(9):e74701. Epub 2013/09/17. doi: 10.1371/journal.pone.0074701. 169. Graifer D, Malygin A, Zharkov DO, Karpova G. Eukaryotic ribosomal protein S3: A constituent of translational machinery and an extraribosomal player in various cellular processes. Biochimie. 2014;99:8-18. Epub 2013/11/19. doi: 10.1016/j.biochi.2013.11.001. 170. Buchan JR. mRNP granules. Assembly, function, and connections with disease. RNA biology. 2014;11(8):1019-30. Epub 2014/12/23. doi: 10.4161/15476286.2014.972208. 171. Ojo KK, Eastman RT, Vidadala R, Zhang Z, Rivas KL, Choi R, et al. A specific inhibitor of PfCDPK4 blocks malaria transmission: chemical-genetic validation. J Infect Dis. 2014;209(2):275-84. Epub 2013/10/15. doi: 10.1093/infdis/jit522. 172. Sebastian S, Brochet M, Collins MO, Schwach F, Jones ML, Goulding D, et al. A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs. Cell Host Microbe. 2012;12(1):9-19. Epub 2012/07/24. doi: 10.1016/j.chom.2012.05.014. 173. Carvalho TG, Doerig C, Reininger L. Nima- and Aurora-related kinases of malaria parasites. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2013;1834(7):1336-45. doi: http://dx.doi.org/10.1016/j.bbapap.2013.02.022. 174. Reininger L, Billker O, Tewari R, Mukhopadhyay A, Fennell C, Dorin-Semblat D, et al. A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites. The Journal of biological chemistry. 2005;280(36):31957-64. Epub 2005/06/23. doi: 10.1074/jbc.M504523200. 175. Barreau C, Paillard L, Mereau A, Osborne HB. Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions. Biochimie. 2006;88(5):515- 25. Epub 2006/02/17. doi: 10.1016/j.biochi.2005.10.011.

115

176. Eisenhaber F, Wechselberger C, Kreil G. The Brix domain protein family -- a key to the ribosomal biogenesis pathway? Trends Biochem Sci. 2001;26(6):345-7. Epub 2001/06/19. 177. Bugner V, Tecza A, Gessert S, Kuhl M. Peter Pan functions independently of its role in ribosome biogenesis during early eye and craniofacial cartilage development in Xenopus laevis. Development. 2011;138(11):2369-78. Epub 2011/05/12. doi: 10.1242/dev.060160. 178. Migeon JC, Garfinkel MS, Edgar BA. Cloning and characterization of peter pan, a novel Drosophila gene required for larval growth. Molecular biology of the cell. 1999;10(6):1733-44. Epub 1999/06/08. 179. Pradel G, Hayton K, Aravind L, Iyer LM, Abrahamsen MS, Bonawitz A, et al. A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito. The Journal of experimental medicine. 2004;199(11):1533-44. 180. Kangwanrangsan N, Tachibana M, Jenwithisuk R, Tsuboi T, Riengrojpitak S, Torii M, et al. A member of the CPW-WPC protein family is expressed in and localized to the surface of developing ookinetes. Malaria journal. 2013;12:129. Epub 2013/04/17. doi: 10.1186/1475-2875-12-129. 181. Dastidar EG, Dayer G, Holland ZM, Dorin-Semblat D, Claes A, Chene A, et al. Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway. BMC biology. 2012;10:5. Epub 2012/02/02. doi: 10.1186/1741-7007-10-5. 182. Tarique M, Ahmad M, Ansari A, Tuteja R. Plasmodium falciparum DOZI, an RNA helicase interacts with eIF4E. Gene. 2013;522(1):46-59. Epub 2013/04/09. doi: 10.1016/j.gene.2013.03.063. 183. Janse CJ, Ramesar J, Waters AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nature protocols. 2006;1(1):346-56. Epub 2007/04/05. doi: 10.1038/nprot.2006.53.

116

184. Umate P, Tuteja N, Tuteja R. Genome-wide comprehensive analysis of human helicases. Communicative & integrative biology. 2011;4(1):118-37. Epub 2011/04/22. doi: 10.4161/cib.4.1.13844. 185. Shankar J, Pradhan A, Tuteja R. Isolation and characterization of Plasmodium falciparum UAP56 homolog: Evidence for the coupling of RNA binding and splicing activity by site- directed mutations. Archives of Biochemistry and Biophysics. 2008;478(2):143-53. doi: http://dx.doi.org/10.1016/j.abb.2008.07.027 186. Ismaili N, Perez-Morga D, Walsh P, Mayeda A, Pays A, Tebabi P, et al. Characterization of a SR protein from Trypanosoma brucei with homology to RNA-binding cis-splicing proteins. Molecular and biochemical parasitology. 1999;102(1):103-15. Epub 1999/09/07. 187. Segref A, Mattaj IW, Ohno M. The evolutionarily conserved region of the U snRNA export mediator PHAX is a novel RNA-binding domain that is essential for U snRNA export. Rna. 2001;7(3):351-60. Epub 2001/05/03. 188. Sugiyama T, Thillainadesan G, Chalamcharla Venkata R, Meng Z, Balachandran V, Dhakshnamoorthy J, et al. Enhancer of Rudimentary Cooperates with Conserved RNA- Processing Factors to Promote Meiotic mRNA Decay and Facultative Heterochromatin Assembly. Molecular cell. 2016;61(5):747-59. doi: 10.1016/j.molcel.2016.01.029. 189. Cunningham DA, Jarra W, Koernig S, Fonager J, Fernandez-Reyes D, Blythe JE, et al. Host immunity modulates transcriptional changes in a multigene family (yir) of rodent malaria. Molecular microbiology. 2005;58(3):636-47. Epub 2005/10/22. doi: 10.1111/j.1365-2958.2005.04840.x. 190. Browning H, Hackney DD. The EB1 homolog Mal3 stimulates the ATPase of the kinesin Tea2 by recruiting it to the microtubule. The Journal of biological chemistry. 2005;280(13):12299-304. Epub 2005/01/25. doi: 10.1074/jbc.M413620200. 191. Lamanna AC, Karbstein K. Nob1 binds the single-stranded cleavage site D at the 3'-end of 18S rRNA with its PIN domain. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(34):14259-64. Epub 2009/08/27. doi: 10.1073/pnas.0905403106.

117

192. Lal K, Delves MJ, Bromley E, Wastling JM, Tomley FM, Sinden RE. Plasmodium male development gene-1 (mdv-1) is important for female sexual development and identifies a polarised plasma membrane during zygote development. International journal for parasitology. 2009;39(7):755-61. Epub 2009/01/13. doi: 10.1016/j.ijpara.2008.11.008. 193. Talman AM, Lacroix C, Marques SR, Blagborough AM, Carzaniga R, Menard R, et al. PbGEST mediates malaria transmission to both mosquito and vertebrate host. Molecular microbiology. 2011;82(2):462-74. Epub 2011/10/01. doi: 10.1111/j.1365- 2958.2011.07823.x. 194. Ponzi M, Siden-Kiamos I, Bertuccini L, Curra C, Kroeze H, Camarda G, et al. Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV- 1/PEG3 protein. Cell Microbiol. 2009;11(8):1272-88. Epub 2009/05/15. doi: 10.1111/j.1462-5822.2009.01331.x. 195. Kariu T, Ishino T, Yano K, Chinzei Y, Yuda M. CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Molecular microbiology. 2006;59(5):1369-79. Epub 2006/02/14. doi: 10.1111/j.1365-2958.2005.05024.x. 196. Ecker A, Bushell ES, Tewari R, Sinden RE. Reverse genetics screen identifies six proteins important for malaria development in the mosquito. Molecular microbiology. 2008;70(1):209-20. Epub 2008/09/03. doi: 10.1111/j.1365-2958.2008.06407.x. 197. Caro F, Ahyong V, Betegon M, DeRisi JL. Genome-wide regulatory dynamics of translation in the asexual blood stages. Elife. 2014;3. doi: 10.7554/eLife.04106. 198. Foth BJ, Zhang N, Chaal BK, Sze SK, Preiser PR, Bozdech Z. Quantitative time-course profiling of parasite and host cell proteins in the human malaria parasite Plasmodium falciparum. Molecular & cellular proteomics : MCP. 2011;10(8):M110.006411. Epub 2011/05/12. doi: 10.1074/mcp.M110.006411. 199. Lee CD, Tu BP. Glucose-Regulated Phosphorylation of the PUF Protein Puf3 Regulates the Translational Fate of Its Bound mRNAs and Association with RNA Granules. Cell Rep. 2015;11(10):1638-50. doi: 10.1016/j.celrep.2015.05.014.

118

200. Solyakov L, Halbert J, Alam MM, Semblat JP, Dorin-Semblat D, Reininger L, et al. Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum. Nat Commun. 2011;2:565. Epub 2011/12/01. doi: 10.1038/ncomms1558. 201. Aravind L, Iyer LM, Anantharaman V. The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism. Genome biology. 2003;4(10):R64. Epub 2003/10/02. doi: 10.1186/gb-2003-4-10-r64. 202. Zhang M, Fennell C, Ranford-Cartwright L, Sakthivel R, Gueirard P, Meister S, et al. The Plasmodium eukaryotic initiation factor-2alpha kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. The Journal of experimental medicine. 2010;207(7):1465-74. Epub 2010/06/30. doi: 10.1084/jem.20091975. 203. Zhang M, Mishra S, Sakthivel R, Rojas M, Ranjan R, Sullivan WJ, Jr., et al. PK4, a eukaryotic initiation factor 2alpha(eIF2alpha) kinase, is essential for the development of the erythrocytic cycle of Plasmodium. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(10):3956-61. Epub 2012/02/23. doi: 10.1073/pnas.1121567109. 204. Friend K, Campbell ZT, Cooke A, Kroll-Conner P, Wickens MP, Kimble J. A conserved PUF- Ago-eEF1A complex attenuates translation elongation. Nature structural & molecular biology. 2012;19(2):176-83. 205. El-Manzalway Y, Munoz EE, Linder SE, Hanovar V. PlasmoSEP: Predicting surface- exposed proteins on the malaria parasite using semisupervised self-training and expert- annotated data. Proteomics. 2016;16(23):2967-2976. 206. Zhang W, Feng D, Li Y, Iida K, McGrath B, et al. PERK EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required for postnatal glucose homeostasis. Cell Metabolism. 2006;4:491–497 207. Senee V, Chelala C, Duchatelet S, Feng D, Blanc H, et al. Mutations in GLIS3 are responsible for a rare syndrome with neonatal diabetes mellitus and congenital hypothyroidism. Nature Genetics. 2006;38:682–687.

119

VITA ELYSE ELAINE MUÑOZ Elyse was born and raised in San Antonio, Texas, and is a huge San Antonio Spurs fan. Growing up in south Texas, she routinely caught frogs, toads, snakes, and other creepy crawlers in her backyard, much to the chagrin of her parents. Recognizing her interest in nature, Elyse was given her first microscope when she was six years old, but hated when anyone suggested she be a scientist. She had big ambitions to be a Broadway actress. Following high school, Elyse attended Arizona State University in Tempe, Arizona on a full-tuition National Hispanic Scholar scholarship. She began her studies in political science, but after her freshman year switched to biology. Her first research experience included work with Gila monsters and venomous snakes, leading to her second, which included work on the effects of atmospheric oxygen on the growth and development of cockroaches and dragonflies. Following completion of her degree, she decided to pursue a PhD at Penn State for two reasons: the amazing Genetics program, and because she wanted to live somewhere cold. She regrets her latter reasoning. Elyse is a proud minority scientist, and seeks to use her unique position to make the term obsolete. She believes in fighting injustices wherever they may be, and does not believe in keeping her mouth shut. After this adventure, she plans to remain in science in one way or another, blending her public speaking skills with her passion for discovering and revealing the underlying wonders of our world.