Exploring the gut microbiota and its potential as a biomarker in gliomas.

Gut microbiome alterations are associated with various cancers including brain tumours such as glioma and glioblastoma. The gut communicates with the brain via a bidirectional pathway known as the gut-brain axis (GBA) which is essential for maintaining homeostasis. The gut microbiota produces many metabolites including short chain fatty acids (SCFAs) and essential amino acids such as glutamate, glutamine, arginine and tryptophan. Through the modulation of these metabolites the gut microbiome is able to regulate several functions of brain cells, immune cells and tumour cells including DNA methylation, mitochondrial function, the aryl hydrocarbon receptor (AhR), T-cell proliferation, autophagy and even apoptosis. Here, we summarise current findings on gut microbiome with respect to brain cancers, an area of research that is widely overlooked. Several studies investigated the relationship between gut microbiota and brain tumours. However, it remains unclear whether the gut microbiome variation is a cause or product of cancer. Subsequently, a biomarker panel was constructed for use as a predictive, prognostic and diagnostic tool with respect to multiple cancers including glioma and glioblastoma multiforme (GBM). This review further presents the intratumoural microbiome, a fascinating microenvironment within the tumour as a possible treatment target that can be manipulated to maximise effectiveness of treatment via personalised therapy. Studies utilising the microbiome as a biomarker and therapeutic strategy are necessary to accurately assess the effectiveness of the gut microbiome as a clinical tool with respect to brain cancers.

Role of microRNAs in COVID-19 with implications for therapeutics.

COVID-19 is a pneumonia-like disease with highly transmittable and pathogenic properties caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects both animals and humans. Although many efforts are currently underway to test possible therapies, there is no specific FDA approved drug against SARS-CoV-2 yet. miRNA-directed gene regulation controls the majority of biological processes. In addition, the development and progression of several human diseases are associated with dysregulation of miRNAs. In this regard, it has been shown that changes in miRNAs are linked to severity of COVID-19 especially in patients with respiratory diseases, diabetes, heart failure or kidney problems. Therefore, targeting these small noncoding-RNAs could potentially alleviate complications from COVID-19. Here, we will review the roles and importance of host and RNA virus encoded miRNAs in COVID-19 pathogenicity and immune response. Then, we focus on potential miRNA therapeutics in the patients who are at increased risk for severe disease.

Hiding in Plain Sight: Formation and Function of Stress Granules During Microbial Infection of Mammalian Cells.

Stress granule (SG) formation is a host cell response to stress-induced translational repression. SGs assemble with RNA-binding proteins and translationally silent mRNA. SGs have been demonstrated to be both inhibitory to viruses, as well as being subverted for viral roles. In contrast, the function of SGs during non-viral microbial infections remains largely unexplored. A handful of microbial infections have been shown to result in host SG assembly. Nevertheless, a large body of evidence suggests SG formation in hosts is a widespread response to microbial infection. Diverse stresses caused by microbes and their products can activate the integrated stress response in order to inhibit translation initiation through phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). This translational response in other contexts results in SG assembly, suggesting that SG assembly can be a general phenomenon during microbial infection. This review explores evidence for host SG formation in response to bacterial, fungal, and protozoan infection and potential functions of SGs in the host and for adaptations of the pathogen.

SCIRT lncRNA Restrains Tumorigenesis by Opposing Transcriptional Programs of Tumor-Initiating Cells.

In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet, how transcriptional regulation of cell-cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle gene expression and increase in self-renewal gene expression are coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long noncoding RNA (lncRNA) that we named Stem Cell Inhibitory RNA Transcript (SCIRT), which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell-cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell-cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing toward repression. These data suggest that the interaction of an lncRNA with EZH2 can alter the affinity of EZH2 for its protein-binding partners to regulate cancer cell state transitions. SIGNIFICANCE: These findings show that a novel lncRNA SCIRT counteracts breast tumorigenesis by opposing transcriptional networks associated with cell cycle and self-renewal.See related commentary by Pardini and Dragomir, p. 535.

A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling.

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems.

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress.

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing.

Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation.

Interrelations between translation and general mRNA degradation in yeast.

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system.

Ways and means of eukaryotic mRNA decay.

Messenger RNA degradation is an important point of control for gene expression. Genome-wide studies on mRNA stability have demonstrated its importance in adaptation and stress response. Most of the key players in mRNA decay appear to have been identified. The study of these proteins brings insight into the mechanism of general and specific targeting of transcripts for degradation. Recruitment and assembly of mRNP complexes enhance and bring specificity to mRNA decay. mRNP complexes can form larger structures that have been found to be ubiquitous in nature. Discovery of P-Bodies, an archetype of this sort of aggregates, has generated interest in the question of where mRNA degrades. This is currently an open question under extensive investigation. This review will discuss in detail the recent developments in the regulation of mRNA decay focusing on yeast as a model system. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Analyzing P-bodies and stress granules in Saccharomyces cerevisiae.

Eukaryotic cells contain at least two types of cytoplasmic RNA-protein (RNP) granules that contain nontranslating mRNAs. One such RNP granule is a P-body, which contains translationally inactive mRNAs and proteins involved in mRNA degradation and translation repression. A second such RNP granule is a stress granule which also contains mRNAs, some RNA binding proteins and several translation initiation factors, suggesting these granules contain mRNAs stalled in translation initiation. In this chapter, we describe methods to analyze P-bodies and stress granules in Saccharomyces cerevisiae, including procedures to determine if a protein or mRNA can accumulate in either granule, if an environmental perturbation or mutation affects granule size and number, and granule quantification methods.

Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

Analyzing P-bodies in Saccharomyces cerevisiae.

Cytoplasmic processing bodies, or P-bodies, are RNA-protein granules found in eukaryotic cells. P-bodies contain non-translating mRNAs and proteins involved in mRNA degradation and translational repression. P-bodies, and the mRNPs within them, have been implicated in mRNA storage, mRNA degradation, and translational repression. The analysis of mRNA turnover often involves the analysis of P-bodies. In this chapter, we describe methods to analyze P-bodies in the budding yeast, Saccharomyces cerevisiae, including procedures to determine whether a protein or mRNA can accumulate in P-bodies, whether an environmental perturbation or mutation affects P-body size and number, and methods to quantify P-bodies.

Computational analysis of miRNA-mediated repression of translation: implications for models of translation initiation inhibition.

The mechanism by which miRNAs inhibit translation has been under scrutiny both in vivo and in vitro. Divergent results have led to the suggestion that miRNAs repress translation by a variety of mechanisms including blocking the function of the cap in stimulating translation. However, these analyses largely only examine the final output of the multistep process of translation. This raises the possibility that when different steps in translation are rate limiting, miRNAs might show different effects on protein production. To examine this possibility, we modeled the process of translation initiation and examined how the effects of miRNAs under different conditions might be explained. Our results suggest that different effects of miRNAs on protein production in separate experiments could be due to differences in rate-limiting steps. This analysis does not rule out that miRNAs directly repress the function of the cap structure, but it demonstrates that the observations used to argue for this effect are open to alternative interpretations. Taking all the data together, our analysis is consistent with the model that miRNAs may primarily repress translation initiation at a late step.

A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits.

Analyses of isolated pre-ribosomes yielded biochemical "snapshots" of the dynamic, nascent 60S and 40S subunits during their path from the nucleolus to the cytoplasm. Here, we present the structure of a pre-60S ribosomal intermediate located in the nucleoplasm. A huge dynein-related AAA-type ATPase (Rea1) and the Rix1 complex (Rix1-Ipi1-Ipi3) are components of an extended (approximately 45 nm long) pre-60S particle. Antibody crosslinking in combination with electron microscopy revealed that the Rea1 localizes to the "tail" region and ribosomal proteins to the "head" region of the elongated "tadpole-like" structure. Furthermore, in vitro treatment with ATP induces dissociation of Rea1 from the pre-60S subunits. Rea1 and the Rix1 complex could mediate ATP-dependent remodeling of 60S subunits and subsequent export from the nucleoplasm to the cytoplasm.

60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm.

60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.