The gene YEF3 function encoding translation elongation factor eEF3 is partially conserved across fungi.

Introduction: Translation is a fundamental process of life. In eukaryotes, the elongation step of translation is highly conserved and is driven by eukaryotic translation elongation factors (eEF)1A and eEF2. A significant variation of the elongation is the activity of eukaryotic elongation factor (eEF) 3 in Saccharomyces cerevisiae encoded by the gene yeast elongation factor (YEF3) with orthologs in all fungal species, a few algae, and some protists. In S. cerevisiae, YEF3 is an essential gene and eEF3 plays a critical role in translation elongation, as it promotes binding of the ternary complex acylated-Transfer RNA (tRNA)-eEF1A-Guanosine-5'-triphosphate (GTP) to the aminoacyl (A) site of the ribosome, the release of uncharged tRNAs after peptide translocation, and ribosome recycling. Even though YEF3 was discovered more than 40 years ago, eEF3 has been characterized almost exclusively in S. cerevisiae. Methods: We undertook an in vivo genetic approach to assess the functional conservation of eEF3 across phylogenetically distant fungal species. Results: We found that eEF3 from Zygosaccharomyces rouxii and Candida glabrata (both belonging to phylum Ascomycota), Ustilago maydis (phylum Basidiomycota), and Gonapodya prolifera (phylum Monoblepharomycota), but not Aspergillus nidulans (phylum Ascomycota), supported the growth of S. cerevisiae lacking the endogenous YEF3 gene. We also proved that eEF3 is an essential gene in the ascomycetes C. glabrata and A. nidulans. Discussion: Given that most existing knowledge on fungal translation has only been obtained from S. cerevisiae, our findings beyond this organism showed variability in the elongation process in Fungi. We also proved that eEF3 is essential in pathogenic fungi, opening the possibility of using eEF3 as a target to fight candidiasis.

The characterization of RNA-binding proteins and RNA metabolism-related proteins in fungal extracellular vesicles.

RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species.

Crosstalk between RNA Metabolism and Cellular Stress Responses during Zika Virus Replication.

Zika virus (ZIKV) is a mosquito-borne virus associated with neurological disorders such as Guillain-Barré syndrome and microcephaly. In humans, ZIKV is able to replicate in cell types from different tissues including placental cells, neurons, and microglia. This intricate virus-cell interaction is accompanied by virally induced changes in the infected cell aimed to promote viral replication as well as cellular responses aimed to counteract or tolerate the virus. Early in the infection, the 11-kb positive-sense RNA genome recruit ribosomes in the cytoplasm and the complex is translocated to the endoplasmic reticulum (ER) for viral protein synthesis. In this process, ZIKV replication is known to induce cellular stress, which triggers both the expression of innate immune genes and the phosphorylation of eukaryotic translation initiation factor 2 (eIF2α), shutting-off host protein synthesis. Remodeling of the ER during ZIKV replication also triggers the unfolded protein response (UPR), which induces changes in the cellular transcriptional landscapes aimed to tolerate infection or trigger apoptosis. Alternatively, ZIKV replication induces changes in the adenosine methylation patterns of specific host mRNAs, which have different consequences in viral replication and cellular fate. In addition, the ZIKV RNA genome undergoes adenosine methylation by the host machinery, which results in the inhibition of viral replication. However, despite these relevant findings, the full scope of these processes to the outcome of infection remains poorly elucidated. This review summarizes relevant aspects of the complex crosstalk between RNA metabolism and cellular stress responses against ZIKV and discusses their possible impact on viral pathogenesis.

Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.

BACKGROUND: RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite. RESULTS: We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress. CONCLUSIONS: The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.

The RNA-binding protein TcUBP1 up-regulates an RNA regulon for a cell surface-associated Trypanosoma cruzi glycoprotein and promotes parasite infectivity.

The regulation of transcription in trypanosomes is unusual. To modulate protein synthesis during their complex developmental stages, these unicellular microorganisms rely largely on post-transcriptional gene expression pathways. These pathways include a plethora of RNA-binding proteins (RBPs) that modulate all steps of the mRNA life cycle in trypanosomes and help organize transcriptomes into clusters of post-transcriptional regulons. The aim of this work was to characterize an RNA regulon comprising numerous transcripts of trypomastigote-associated cell-surface glycoproteins that are preferentially expressed in the infective stages of the human parasite Trypanosoma cruzi. In vitro and in vivo RNA-binding assays disclosed that these glycoprotein mRNAs are targeted by the small trypanosomatid-exclusive RBP in T. cruzi, U-rich RBP 1 (TcUBP1). Overexpression of a GFP-tagged TcUBP1 in replicative parasites resulted in >10 times up-regulated expression of transcripts encoding surface proteins and in changes in their subcellular localization from the posterior region to the perinuclear region of the cytoplasm, as is typically observed in the infective parasite stages. Moreover, RT-quantitative PCR analysis of actively translated mRNAs by sucrose cushion fractionation revealed an increased abundance of these target transcripts in the polysome fraction of TcUBP1-induced samples. Because these surface proteins are involved in cell adherence or invasion during host infection, we also carried out in vitro infections with TcUBP1-transgenic trypomastigotes and observed that TcUBP1 overexpression significantly increases parasite infectivity. Our findings provide evidence for a role of TcUBP1 in trypomastigote stage-specific gene regulation important for T. cruzi virulence.

DEAD/DExH-Box RNA Helicases in Selected Human Parasites.

DEAD/DExH-box RNA helicases catalyze the folding and remodeling of RNA molecules in prokaryotic and eukaryotic cells, as well as in many viruses. They are characterized by the presence of the helicase domain with conserved motifs that are essential for ATP binding and hydrolysis, RNA interaction, and unwinding activities. Large families of DEAD/DExH-box proteins have been described in different organisms, and their role in all molecular processes involving RNA, from transcriptional regulation to mRNA decay, have been described. This review aims to summarize the current knowledge about DEAD/DExH-box proteins in selected protozoan and nematode parasites of medical importance worldwide, such as Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Giardia lamblia, Entamoeba histolytica, and Brugia malayi. We discuss the functional characterization of several proteins in an attempt to understand better the molecular mechanisms involving RNA in these pathogens. The current data also highlight that DEAD/DExH-box RNA helicases might represent feasible drug targets due to their vital role in parasite growth and development.