Eukaryotic Research Articles

Developing skills for a healthy lifestyle by using interactive methods in teaching Biology and Health Education

Abstract The aim of the article is to present the results of using interactive methods in the teaching of Biology and Health Education to 7th grade students for developing skills for a healthy lifestyle. The discussion is based on the topics: “The role of prokaryotic organisms in nature and their importance for man”, “The role of unicellular eukaryotic organisms in nature and their importance for man”, and “Talus plants. Seaweed”. The discussion is supported by fragments of lessons in which interactive methods are used – the method of associations through creating mind maps, discussion, case study, and role play. As a result of using of interactive methods, students develop key competencies for maintaining their own health and the health of society as a whole.

Comparative genomics reveals the evolutionary history of the unicellular eukaryote class Litostomatea and its adaptive evolution based on biochemical metabolic capacity

Abstract Ciliated protists are unicellular eukaryotic organisms characterized by their morphological diversity, ubiquitous distribution, and the important roles they play in a wide range of biological studies. The class Litostomatea is a morphologically diverse ciliate group that comprises hundreds of free-living and endosymbiotic species. Here, we sequenced 14 predatory litostomateans, i.e. 12 haptorians and two rhynchostomatians. A comparative analysis was performed with other published omics’ data on litostomateans. Our first phylogenomic analysis of litostomateans showed the monophyly of the subclasses Trichostomatia and Rhynchostomatia, the non-monophyly of the subclass Haptoria, and the monophyly of all orders and families that were analysed. Evolutionary history analysis suggested that Litostomatea diverged during the Late Neoproterozoic, the family Chaeneidae was the earliest diverging haptorian lineage, and the Rhynchostomatia probably separated from the order Lacrymariida (subclass Haptoria) during the Early Palaeozoic. Stop codon usage analysis of 28 litostomateans showed that they use TAA as the biased stop codon and reassign the other two stop codons (TAG and TGA) to code for amino acids. In addition, the preferred codons in the 14 newly sequenced litostomateans are strongly biased towards A/U bases in the third position, very probably due to the comparatively low GC content. Genes encoding carbohydrate-active enzymes (CAZymes) are more diversified in the endosymbiotic Trichostomatia than in the free-living predatory Rhynchostomatia and Haptoria, suggesting that trichostomes have the strongest capability of carbohydrate utilization. Notably, we found that three free-living litostomateans (Didinium sp.1, Myriokaryon sp., and Apodileptus visscheri) exhibit substantial differences from other free-living ciliates in terms of their number of CAZymes. Considering the potency and versatility of CAZymes in the degradation and biotransformation of carbohydates, we propose that the multifarious CAZymes in these three ciliates could be a survival strategy for nutrient acquisition and niche adaptation. Finally, the functional annotation of significantly expanded gene families in these three ciliates revealed their vigorous potency in biochemical metabolism. These findings will facilitate wider omic-scale phylogenetic analyses of Litostomatea and deepen our understanding of this group from an evolutionary standpoint.

Mitochondrion-to-nucleus communication mediated by RNA export: a survey of potential mechanisms and players across eukaryotes.

The nucleus interacts with the other organelles to perform essential functions of the eukaryotic cell. Mitochondria have their own genome and communicate back to the nucleus in what is known as mitochondrial retrograde response. Information is transferred to the nucleus in many ways, leading to wide-ranging changes in nuclear gene expression and culminating with changes in metabolic, regulatory or stress-related pathways. RNAs are emerging molecules involved in this signalling. RNAs encode precise information and are involved in highly target-specific signalling, through a wide range of processes known as RNA interference. RNA-mediated mitochondrial retrograde response requires these molecules to exit the mitochondrion, a process that is still mostly unknown. We suggest that the proteins/complexes translocases of the inner membrane, polynucleotide phosphorylase, mitochondrial permeability transition pore, and the subunits of oxidative phosphorylation complexes may be responsible for RNA export.

NADPH oxidase 5: Where are we now and which way to proceed?

Since the incorporation of mitochondria in early eukaryotes cells struggle to keep the deleterious effects of reactive oxygen species (ROS), mainly originating from the respiratory chain, at bay. Evolutionary adaptation to ROS burden went so far that by acting as messenger and effector molecules, ROS became important in maintaining homeostasis. The evolutionary success of this phenomenon is underscored by the arising of professional ROS-generating enzymes, namely the family of NADPH oxidases (NOXes). NOXes, by shaping ROS levels at different subcellular locations and in extracellular space, are involved in such fundamental functions as proliferation, differentiation, apoptosis, host defense, fertilization, and hormone biosynthesis. NOX5, being a calcium-regulated professional ROS source exerts its function at the crossroad of these two fundamental but potentially deleterious intracellular signaling pathways (i.e. Ca2+ and ROS). The expression of NOX5 in the adult human body under unchallenged conditions is restricted to very few sites, among which the two major tissue groups are genital organs (mainly testis) and immune tissues (mainly spleen). In cases of increased cellular proliferation and protein synthesis (e.g., diverse tumors, cultured primary cells, or sites of tissue damage) the expression and activity of NOX5 is often upregulated in various tissues. This and the evolutionary conserved nature of NOX5 would imply a very fundamental role for this enzyme, but intriguingly the genomes of rodents essentially lack the NOX5 gene. The latter fact had been a major obstacle in determining the physiological roles of NOX5 in normal tissues until the very recent generation of a NOX5-deficient rabbit model.

Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases

An organism’s genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5′ to 3′ direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e.,"cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Spliceosomal snRNAs, the Essential Players in pre-mRNA Processing in Eukaryotic Nucleus: From Biogenesis to Functions and Spatiotemporal Characteristics.

Spliceosomal small nuclear RNAs (snRNAs) are a fundamental class of non-coding small RNAs abundant in the nucleoplasm of eukaryotic cells, playing a crucial role in splicing precursor messenger RNAs (pre-mRNAs). They are transcribed by DNA-dependent RNA polymerase II (Pol II) or III (Pol III), and undergo subsequent processing and 3' end cleavage to become mature snRNAs. Numerous protein factors are involved in the transcription initiation, elongation, termination, splicing, cellular localization, and terminal modification processes of snRNAs. The transcription and processing of snRNAs are regulated spatiotemporally by various mechanisms, and the homeostatic balance of snRNAs within cells is of great significance for the growth and development of organisms. snRNAs assemble with specific accessory proteins to form small nuclear ribonucleoprotein particles (snRNPs) that are the basal components of spliceosomes responsible for pre-mRNA maturation. This article provides an overview of the biological functions, biosynthesis, terminal structure, and tissue-specific regulation of snRNAs.

The Triterpenoid MOMORDIN-Ic Inhibits HCMV by Preventing the Initiation of Gene Expression in Eukaryotic Cells.

Human cytomegalovirus (HCMV) primary infection, re-infection, and reactivation from latency cause morbidity in immune-compromised patients. Consequently, potential therapeutic strategies remain of interest for the treatment of infection. Naturally occurring triterpenoids derived from plants have been demonstrated to have anti-viral activity, although their precise mechanisms of action are not always fully understood. Here, we investigate the activity of Mormordin Ic (Mc) and demonstrate that it is potently anti-viral against HCMV. Through investigation of the mechanistic basis of this anti-viral activity, we identify that it is inhibitory to both viral and host gene expression, and to highly induced genes in particular. We go on to observe that Mc impacts on RNA Pol II activity and, specifically, reduces the occupancy of elongating RNA Pol II at a viral promoter. Next, we demonstrate that Mc is inhibitory to HCMV reactivation, and in doing so identify that it has greater activity against the canonical major immediate early promoter compared to the alternative ip2 promoter located downstream. Finally, we see evidence of RNA Pol II occupancy at the ip2 promoter in undifferentiated myeloid cells. Thus, Mc is potently anti-viral and a potential tool to probe the activity of multiple promoters considered important for controlling HCMV reactivation.

Yeast Npl3 regulates replicative senescence outside of TERRA R-loop resolution and co-transcriptional processing

Eukaryotic cells without telomerase experience progressively shorter telomeres with each round of cell division until cell cycle arrest is initiated, leading to replicative senescence. When yeast TLC1, which encodes the RNA template of telomerase, is deleted, senescence is accompanied by increased expression of TERRA (non-coding telomere repeat-containing RNA). Deletion of Npl3, an RNA-processing protein with telomere maintenance functions, accelerates senescence in tlc1Δ cells and significantly increases TERRA levels. Using genetic approaches, we set out to determine how Npl3 is involved in regulating TERRA expression and maintaining telomere homeostasis. Even though Npl3 regulates hyperrecombination, we found that Npl3 does not help resolve RNA:DNA hybrids formed during TERRA synthesis in the same way as RNase H1 and H2. Furthermore, Rad52 is still required for cells to escape senescence by telomere recombination in the absence of Npl3. Npl3 also works separately from the THO/TREX pathway for processing nascent RNA for nuclear export. However, deleting Dot1, a histone methyltransferase involved in tethering telomeres to the nuclear periphery, rescued the accelerated senescence phenotype of npl3Δ cells. Thus, our study suggests that Npl3 plays an additional role in regulating cellular senescence outside of RNA:DNA hybrid resolution and co-transcriptional processing.

Mammalian START-like phosphatidylinositol transfer proteins – Physiological perspectives and roles in cancer biology

PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases – including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.

Cytosolic N-terminal formyl-methionine deformylation derives cancer stem cell features and tumor progression

Eukaryotic cells can synthesize formyl-methionine (fMet)-containing proteins not only in mitochondria but also in the cytosol to some extent. Our previous study revealed substantial upregulation of N-terminal (Nt)-fMet-containing proteins in the cytosol of SW480 colorectal cancer cells. However, the functional and pathophysiological implications remain unclear. Here, we demonstrated that removal of the Nt-formyl moiety of Nt-fMet-containing proteins (via expressing Escherichia coli PDF peptide deformylase) resulted in a dramatic increase in the proliferation of SW480 colorectal cancer cells. This proliferation coincided with the acquisition of cancer stem cell features, including reduced cell size, enhanced self-renewal capacity, and elevated levels of the cancer stem cell surface marker CD24 and pluripotent transcription factor SOX2. Furthermore, deformylation of Nt-fMet-containing proteins promoted the tumorigenicity of SW480 colorectal cancer cells in an in vivo xenograft mouse model. Taken together, these findings suggest that cytosolic deformylation has a tumor-enhancing effect, highlighting its therapeutic potential for cancer treatment.

Genes divided according to the relative position of the longest intron show increased representation in different KEGG pathways

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes.

Novel Z-DNA binding domains in giant viruses

Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.

The Roles of the Anthraquinone Parietin in the Tolerance to Desiccation of the Lichen Xanthoria parietina: Physiology and Anatomy of the Pale and Bright-Orange Thalli.

Lichens are symbiotic organisms that effectively survive in harsh environments, including arid regions. Maintaining viability with an almost complete loss of water and the rapid restoration of metabolism during rehydration distinguishes lichens from most eukaryotic organisms. The lichen Xanthoria parietina is known to have high stress tolerance, possessing diverse defense mechanisms, including the presence of the bright-orange pigment parietin. While several studies have demonstrated the photoprotective and antioxidant properties of this anthraquinone, the role of parietin in the tolerance of lichens to desiccation is not clear yet. Thalli, which are exposed to solar radiation and become bright orange, may require enhanced desiccation tolerance. Here, we showed differences in the anatomy of naturally pale and bright-orange thalli of X. parietina and visualized parietin crystals on the surface of the upper cortex. Parietin was extracted from bright-orange thalli by acetone rinsing and quantified using HPLC. Although acetone rinsing did not affect PSII activity, thalli without parietin had higher levels of lipid peroxidation and a lower membrane stability index in response to desiccation. Furthermore, highly pigmented thalli possess thicker cell walls and, according to thermogravimetric analysis, higher water-holding capacities than pale thalli. Thus, parietin may play a role in desiccation tolerance by stabilizing mycobiont membranes, providing an antioxidative defense, and changing the morphology of the upper cortex of X. parietina.

Extracellular signal-regulated protein kinase 5 modulates the spindle assembly to coordinate the oocyte meiotic maturation

Extracellular signal-regulated protein kinase 5 (Erk5), a member of the mitogen-activated protein kinase (MAPK) family, is ubiquitously expressed in all eukaryotic cells and is implicated in the various mitotic processes such as cell survival, proliferation, migration, and differentiation. However, the potential functional roles of Erk5 in oocyte meiosis have not been fully determined. In this study, we document that ERK5 participates in the meiotic maturation of mouse oocytes by regulating the spindle assembly to ensure the meiotic progression. We unexpectedly found that phosphorylated ERK5 was localized in the spindle pole region at metaphase I and II stages by immunostaining analysis. Inhibition of ERK5 activity using its specific inhibitor XMD8-92 dramatically reduced the incidence of first polar body extrusion. In addition, inhibition of ERK5 evoked the spindle assembly checkpoint to arrest oocytes at metaphase I stage by impairing the spindle assembly, chromosome alignment and kinetochore-microtubule attachment. Mechanically, over-strengthened microtubule stability was shown to disrupt the microtubule dynamics and thus compromise the spindle assembly in ERK5-inhibited oocytes. Conversely, overexpression of ERK5 caused decreased level of acetylated α-tubulin and spindle defects. Collectively, we conclude that ERK5 plays an important role in the oocyte meiotic maturation by regulating microtubule dynamics and spindle assembly.

A comprehensive review of soybean RNL and TIR domain proteins.

Both prokaryotic and eukaryotic organisms use the nucleotide-binding domain/leucine-rich repeat (NBD/LRR)-triggered immunity (NLR-triggered immunity) signaling pathway to defend against pathogens. Plant NLRs are intracellular immune receptors that can bind to effector proteins secreted by pathogens. Dicotyledonous plants express a type of NLR known as TIR domain-containing NLRs (TNLs). TIR domains are enzymes that catalyze the production of small molecules that are essential for immune signaling and lead to plant cell death. The activation of downstream TNL signaling components, such as enhanced disease susceptibility 1 (EDS1), phytoalexin deficient 4 (PAD4), and senescence-associated gene 101 (SAG101), is facilitated by these small molecules. Helper NLRs (hNLRs) and the EDS1-PAD4/SAG101 complex associate after activation, causing the hNLRs to oligomerize, translocate to the plasma membrane (PM), and produce cation-selective channels. According to a recent theory, cations enter cells through pores created by oligomeric hNLRs and trigger cell death. Occasionally, TNLs can self-associate to create higher-order oligomers. Here, we categorized soybean TNLs based on the protein domains that they possess. We believe that TNLs may help soybean plants effectively fight pathogens by acting as a source of genetic resistance. In summary, the purpose of this review is to elucidate the range of TNLs that are expressed in soybean.

An organometallic hybrid antibiotic of metronidazole with a Gold(I) N-Heterocyclic Carbene overcomes metronidazole resistance in Clostridioides difficile.

Antimicrobial resistance (AMR) has been emerging as a major global health threat and calls for the development of novel drug candidates. Metal complexes have been demonstrating high efficiency as antibacterial agents that differ substantially from the established types of antibiotics in their chemical structures and their mechanism of action. One strategy to exploit this potential is the design of metal-based hybrid organometallics that consist of an established antibiotic and a metal-based warhead that contributes an additional mechanism of action different from that of the parent antibiotic. In this communication, we describe the organometallic hybrid antibiotic 2c, in which the drug metronidazole is connected to a gold(I) N-heterocyclic carbene warhead that inhibits bacterial thioredoxin reductase (TrxR). Metronidazole can be used for the treatment with the obligatory anaerobic pathogen Clostridioides difficile (C. difficile), however, resistance to the drug hampers its clinical success. The gold organometallic conjugate 2c was an efficient inhibitor of TrxR and it was inactive or showed only minor effects against eucaryotic cells and bacteria grown under aerobic conditions. In contrast, a strong antibacterial effect was observed against both metronidazole-sensitive and -resistant strains of C. difficile. This report presents a proof-of-concept that the design of metal-based hybrid antibiotics can be a viable approach to efficiently tackle AMR.

Engineering circular RNA for molecular and metabolic reprogramming.

The role of messenger RNA (mRNA) in biological systems is extremely versatile. However, it's extremely short half-life poses a fundamental restriction on its application. Moreover, the translation efficiency of mRNA is also limited. On the contrary, circular RNAs, also known as circRNAs, are a common and stable form of RNA found in eukaryotic cells. These molecules are synthesized via back-splicing. Both synthetic circRNAs and certain endogenous circRNAs have the potential to encode proteins, hence suggesting the potential of circRNA as a gene expression machinery. Herein, we aim to summarize all engineering aspects that allow exogenous circular RNA (circRNA) to prolong the time that proteins are expressed from full-length RNA signals. This review presents a systematic engineering approach that have been devised to efficiently assemble circRNAs and evaluate several aspects that have an impact on protein production derived from. We have also reviewed how optimization of the key components of circRNAs, including the topology of vector, 5' and 3' untranslated sections, entrance site of the internal ribosome, and engineered aptamers could be efficiently impacting the translation machinery for molecular and metabolic reprogramming. Collectively, molecular and metabolic reprogramming present a novel way of regulating distinctive cellular features, for instance growth traits to neoplastic cells, and offer new possibilities for therapeutic inventions.

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion.

Eukaryotic cells have developed intricate mechanisms for biomolecule transport, particularly in stressful conditions. This interdisciplinary study delves into unconventional protein secretion (UPS) pathways activated during starvation, facilitating the export of proteins bypassing most of the components of the classical secretory machinery. Specifically, we focus on the underexplored mechanisms of the GRASP's role in UPS, particularly in biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results show that liquid-liquid phase separation (LLPS) plays a key role in the coacervation of Grh1, the GRASP yeast homologue, under starvation-like conditions. This association seems a precursor to the Compartment for Unconventional Protein Secretion (CUPS) biogenesis. Grh1's self-association is regulated by electrostatic, hydrophobic, and hydrogen-bonding interactions. Importantly, our study demonstrates that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations. Additionally, we explore how the coacervate liquid-to-solid transition could impact cells' ability to return to normal post-stress states. Our findings offer insights into intracellular protein dynamics and cell adaptive responses to stress.

Measurements of soil protist richness and community composition are influenced by primer pair, annealing temperature, and bioinformatics choices.

Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding.IMPORTANCEEcosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.