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The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.

In response to the host environment, Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host temperature and oxidative stress. This reprogramming is primarily driven through the Gcn2-mediated repression of translation initiation and Ccr4-mediated removal of abundant pro-growth mRNAs from the translating pool. Here we investigate the contributions of these two pathways to the translational response to stress, and show that the response to oxidative stress is primarily driven by Gcn2 whereas temperature and oxidative stress both require Ccr4. Temperature stress, but not oxidative stress, result in an increase in RNase I resistant disome. Further, eIF2α phosphorylation varies in response to the type and magnitude of stress, yet all tested conditions induce translation of integrated stress response (ISR) transcription factor Gcn4, but not necessarily the Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress in C. neoformans. Together this study identifies the differential response to host-relevant stressors in an environmental fungus which can adapt to the environment inside the human host.

SummaryThe evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5Δ mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5Δ mutant showed that Rmt5 is critical for the homeostasis of eIF2α and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5Δ clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5Δ phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins.SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

The fungal pathogen Cryptococcus neoformans relies on post-transcriptional mechanisms of gene regulation to adapt to stressors it encounters in the human host, such as oxidative stress and nutrient limitation. The kinase Gcn2 regulates translation in response to stress by phosphorylating the initiation factor eIF2, and it is a crucial factor in withstanding oxidative stress in C. neoformans, and amino acid limitation in many fungal species. However, little is known about the role of Gcn2 in nitrogen limitation in C. neoformans. In this study, we demonstrate that Gcn2 is required for C. neoformans to utilize methionine as a source of nitrogen, and that the presence of methionine as a sole nitrogen source induces eIF2 phosphorylation. The stress imposed by methionine leads to an oxidative stress response at both the levels of transcription and translation, as seen through polysome profiling as well as increased abundance of select oxidative stress response transcripts. The transcription factor Gcn4 is also required for methionine utilization and oxidative stress resistance, and RT-qPCR data suggests that it regulates expression of certain transcripts in response to oxidative stress. The results of this study suggest a connection between nitrogen metabolism and oxidative stress in C. neoformans that is mediated by Gcn4, possibly indicating the presence of a compound stress response in this clinically important fungal pathogen.

Cryptococcus neoformans is a devastating opportunistic fungal pathogen. It mostly impacts people in an immunocompromised state, such as people living with HIV/AIDS and following organ transplantation. Macrophages, in addition to being a major cellular reservoir of HIV-1, represent a unique niche in which both C. neoformans and HIV-1 can coinhabit in the course of natural infection. Here, we report the observation that HIV-1 infection of THP-1 macrophages increases the rate at which they phagocytose C. neoformans cells. We investigated the tumor necrosis factor alpha (TNF-α) signaling and nuclear factor kappa B (NF-κB) activation in human monocyte-derived macrophages infected with HIV-1 alone, as well as those coinfected with HIV-1 and C. neoformans. Our findings showed that while HIV-1 infection alone upregulates TNF-α production and activates NF-κB signaling, C. neoformans coinfection drastically and rapidly dampens this proinflammatory response. These data suggest an antagonism between two important human pathogens during coinfection of macrophages. IMPORTANCE Fungal infections are one of the leading causes of death for people who live with HIV/AIDS. Even though these pathogens are independently well studied, it is still enigmatic how coinfection with HIV-1 and C. neoformans alters gene expression and cellular processes, especially in clinically relevant cell types. Understanding the interplay between these two pathogens is especially critical because C. neoformans mortality largely depends on the host’s immunocompromised state during viral infection. Studying this coinfection is challenging since HIV-1 only infects human cells, and the modified murine HIV-1 virus does not reproduce the clinical landmarks of HIV-1 infection or AIDS in mice. Our observations shed light on how these two pathogens trigger opposing trends in TNF-α and NF-κB signaling in human monocyte-derived macrophages.

The fungal pathogen Cryptococcus neoformans relies on post-transcriptional mechanisms of gene regulation to adapt to stressors it encounters in the human host, such as oxidative stress and nutrient limitation. The kinase Gcn2 regulates translation in response to stress by phosphorylating the initiation factor eIF2, and it is a crucial factor in withstanding oxidative stress in C. neoformans, and amino acid limitation in many fungal species. However, little is known about the role of Gcn2 in nitrogen limitation in C. neoformans. In this study, we demonstrate that Gcn2 is required for C. neoformans to utilize methionine as a source of nitrogen, and that the presence of methionine as a sole nitrogen source induces eIF2 phosphorylation. The stress imposed by methionine leads to an oxidative stress response at both the levels of transcription and translation, as seen through polysome profiling as well as increased abundance of select oxidative stress response transcripts. The transcription factor Gcn4 is also required for methionine utilization and oxidative stress resistance, and RT-qPCR data suggests that it regulates expression of certain transcripts in response to oxidative stress. The results of this study suggest a connection between nitrogen metabolism and oxidative stress in C. neoformans that is mediated by Gcn4, possibly indicating the presence of a compound stress response in this clinically important fungal pathogen.

Echinocandins have been on the market for 20 years, yet they are the newest class of antifungal drugs. The human fungal pathogenCryptococcus neoformansis intrinsically resistant to the echinocandin antifungal drug caspofungin, which targets theβ-1,3-glucan synthase encoded by theFKS1. Analysis of aC. neoformans puf4Δ mutant, lacking the pumilio/FBF RNA binding protein family member Puf4, revealed exacerbated caspofungin resistance. In contrast, overexpression ofPUF4resulted in caspofungin sensitivity. TheFKS1mRNA contains three Puf4-binding elements (PBEs) in its 5’ untranslated region. Puf4 binds with specificity to this region of theFKS1. TheFKS1mRNA was destabilized in thepuf4Δ mutant, and the abundance of theFKS1mRNA was reduced compared to wild type, suggesting that Puf4 is a positive regulatorFKS1mRNA stability. In addition toFKS1, the abundance of additional cell wall biosynthesis genes, including chitin synthases (CHS3,CHS4,CHS6) and deacetylases (CDA1,CDA2,CDA3) as well as aβ-1,6-glucan synthase gene (SKN1) was regulated by Puf4 during a caspofungin time course. The use of fluorescent dyes to quantify cell wall components revealed that thepuf4Δ mutant had increased chitin content, suggesting a cell wall composition that is less reliant onβ-1,3-glucan. Overall, our findings suggest a mechanism by which caspofungin resistance, and more broadly, cell wall biogenesis, is regulated post-transcriptionally by Puf4.ImportanceCryptococcus neoformansis an environmental fungus that causes pulmonary and central nervous system infections. It is also responsible for 15% of AIDS-related deaths. A major contributor to the high morbidity and mortality statistics is the lack of safe and effective antifungal therapies, especially in resource-poor settings. Yet, antifungal drug development has stalled in the pharmaceutical industry. Therefore, it is of importance to understand the mechanism by whichC. neoformansis resistant to caspofungin in order to design adjunctive therapies to potentiate its activity toward this important pathogen.

The insect-transmitted protozoan parasite Trypanosoma cruzi experiences changes in nutrient availability and rate of flux through different metabolic pathways across its life cycle. The species encompasses much genetic diversity of both the nuclear and mitochondrial genomes among isolated strains. The genetic or expression variation of both genomes are likely to impact metabolic responses to environmental stimuli, and even steady state metabolic function, among strains. To begin formal characterization these differences, we compared aspects of metabolism between genetically similar strains CL Brener and Tulahuen with less similar Esmeraldo and Sylvio X10 strains in a culture environment. Epimastigotes of all strains took up glucose at similar rates. However, the degree of medium acidification that could be observed when glucose was absent from the medium varied by strain, indicating potential differences in excreted metabolic byproducts. Our main focus was differences related to electron transport chain function. We observed differences in ATP-coupled respiration and maximal respiratory capacity, mitochondrial membrane potential, and mitochondrial morphology between strains, despite the fact that abundances of two nuclear-encoded proteins of the electron transport chain are similar between strains. RNA sequencing reveals strain-specific differences in abundances of mRNAs encoding proteins of the respiratory chain but also other metabolic processes. From these differences in metabolism and mitochondrial phenotypes we have generated tentative models for the differential metabolic fluxes or differences in gene expression that may underlie these results.