Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation ...initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.
Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit ...membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.
Differential gene expression analysis may discover a set of genes too large to easily investigate, so a means of ranking genes by biological interest level is desired. p values are frequently abused ...for this purpose. As an alternative, we propose a method of ranking by confidence bounds on the log fold change, based on the previously developed TREAT test. These confidence bounds provide guaranteed false discovery rate and false coverage-statement rate control. When applied to a breast cancer dataset, the top-ranked genes by Topconfects emphasize markedly different biological processes compared to the top-ranked genes by p value.
Mitochondrial complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly ...factors. The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, and COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly.
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•The MCIA complex is required for stability of mtDNA-encoded ND2•Assembly factors show a hierarchy of stability centered on ACAD9•TMEM186 interacts strongly with newly synthesized ND3•COA1 interacts with newly translated ND2 and is dispensable for complex IV assembly
Formosa et al. investigate the function of the MCIA complex in complex I assembly. They demonstrate the requirement of individual components for the formation of complex I intermediates and assembly of the final enzyme. Finally, they characterize the involvement of TMEM186 and COA1 in this process.
To fight infections, macrophages undergo a metabolic shift whereby increased glycolysis fuels antimicrobial inflammation and killing of pathogens. Here we demonstrate that the pathogen Candida ...albicans turns this metabolic reprogramming into an Achilles' heel for macrophages. During Candida-macrophage interactions intertwined metabolic shifts occur, with concomitant upregulation of glycolysis in both host and pathogen setting up glucose competition. Candida thrives on multiple carbon sources, but infected macrophages are metabolically trapped in glycolysis and depend on glucose for viability: Candida exploits this limitation by depleting glucose, triggering rapid macrophage death. Using pharmacological or genetic means to modulate glucose metabolism of host and/or pathogen, we show that Candida infection perturbs host glucose homeostasis in the murine candidemia model and demonstrate that glucose supplementation improves host outcomes. Our results support the importance of maintaining glucose homeostasis for immune cell survival during Candida challenge and for host survival in systemic infection.
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•The macrophage Warburg effect becomes a liability during microbial challenge•In vitro, Candida competes for glucose, triggering massive macrophage death•In vivo, Candida infection promotes disruption of host glucose homeostasis•A glucose-rich diet improves host outcomes in systemic fungal infection
Glucose is essential for innate immune cells to mount effective anti-fungal responses. Tucey et al. show that, for infected macrophages, dependence on glucose becomes their downfall: the human fungal pathogen Candida albicans rapidly consumes glucose, causing macrophages to die. In mice, maintaining host glucose homeostasis is important to prevent life-threatening fungal infection.
Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome ...with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation. Importantly, it does not monitor multiple substeps of initiation and termination that involve complexes between the small ribosomal subunit (SSU) and mRNA. Here we describe a related method, termed 'translation complex profile sequencing' (TCP-seq), that is uniquely capable of recording positions of any type of ribosome-mRNA complex transcriptome-wide. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into complexes involving either the full ribosome or the SSU. The footprints derived from each type of complex are then deep-sequenced separately, generating native distribution profiles during the elongation, as well as the initiation and termination stages of translation. We provide the full TCP-seq protocol for Saccharomyces cerevisiae liquid suspension culture, including a data analysis pipeline. The protocol takes ∼3 weeks to complete by a researcher who is well acquainted with standard molecular biology techniques and who has some experience in ultracentrifugation and the preparation of RNA sequencing (RNA-seq) libraries. Basic Bash and UNIX/Linux command skills are required to use the bioinformatics tools provided.
Fungal pathogens overcome antifungal drug therapy by classic resistance mechanisms, such as increased efflux or changes to the drug target. However, even when a fungal strain is susceptible, trailing ...or persistent microbial growth in the presence of an antifungal drug can contribute to therapeutic failure. This trailing growth is caused by adaptive physiological changes that enable the growth of a subpopulation of fungal cells in high drug concentrations, in what is described as drug tolerance. Mechanistically, antifungal drug tolerance is incompletely understood. Here we report that the transcriptional activator Rpn4 is important for drug tolerance in the human fungal pathogen Candida albicans. Deletion of RPN4 eliminates tolerance to the commonly used antifungal drug fluconazole. We defined the mechanism and show that Rpn4 controls fluconazole tolerance via two target pathways. First, Rpn4 activates proteasome gene expression, which enables sufficient proteasome capacity to overcome fluconazole-induced proteotoxicity and the accumulation of ubiquitinated proteins targeted for degradation. Consistently, inhibition of the proteasome with MG132 eliminates fluconazole tolerance and resistance, and phenocopies the rpn4Δ/Δ mutant for loss of tolerance. Second, Rpn4 is required for wild type expression of the genes required for the synthesis of the membrane lipid ergosterol. Our data indicates that this function of Rpn4 is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. Based on our findings, we propose that Rpn4 is a central hub for fluconazole tolerance in C. albicans by coupling the regulation of protein homeostasis (proteostasis) and lipid metabolism to overcome drug-induced proteotoxicity and membrane stress.
Control of poly(A) tail length can affect translation and stability of eukaryotic mRNAs. Although well established for individual cases, it was not known to what extent this type of adjustable gene ...control is used to shape expression of eukaryotic transcriptomes. Here we report on microarray-based measurements of mRNA poly(A) tail lengths and association with the poly(A)-binding protein Pab1 in S. cerevisiae, revealing extensive correlation between tail length and other physical and functional mRNA characteristics. Gene ontology analyses and further directed experiments indicate coregulation of tail length on functionally and cytotopically related mRNAs to coordinate cell-cycle progression, ribosome biogenesis, and retrotransposon expression. We show that the 3'-untranslated region drives transcript-specific adenylation control and translational efficiency of multiple mRNAs. Our findings suggest a wide-spread interdependence between 3'-untranslated region-mediated poly(A) tail length control, Pab1 binding, and mRNA translation in budding yeast. They further provide a molecular explanation for deadenylase function in the cell cycle and suggest additional cellular processes that depend on control of mRNA polyadenylation.
Abstract
Differentiation of neural progenitor cells into mature neuronal phenotypes relies on extensive temporospatial coordination of mRNA expression to support the development of functional brain ...circuitry. Cleavage and polyadenylation of mRNA has tremendous regulatory capacity through the alteration of mRNA stability and modulation of microRNA (miRNA) function, however the extent of utilization in neuronal development is currently unclear. Here, we employed poly(A) tail sequencing, mRNA sequencing, ribosome profiling and small RNA sequencing to explore the functional relationship between mRNA abundance, translation, poly(A) tail length, alternative polyadenylation (APA) and miRNA expression in an in vitro model of neuronal differentiation. Differential analysis revealed a strong bias towards poly(A) tail and 3′UTR lengthening during differentiation, both of which were positively correlated with changes in mRNA abundance, but not translation. Globally, changes in miRNA expression were predominantly associated with mRNA abundance and translation, however several miRNA–mRNA pairings with potential to regulate poly(A) tail length were identified. Furthermore, 3′UTR lengthening was observed to significantly increase the inclusion of non-conserved miRNA binding sites, potentially enhancing the regulatory capacity of these molecules in mature neuronal cells. Together, our findings suggest poly(A) tail length and APA function as part of a rich post-transcriptional regulatory matrix during neuronal differentiation.
Graphical Abstract
Graphical Abstract
Microbes are exposed to nutritional and stress challenges in their environmental and host niches. To rise to these challenges, they regulate transcriptional programs that enable cellular adaptation. ...For instance, metabolite concentrations regulate post-translational modifications of chromatin, such as histone acetylation. In this way, metabolic signals are integrated with transcription. Over the last decade, several histone acylations have been discovered, including histone crotonylation. Their roles in microbial biology, environmental adaptation, and microbe-host interactions are incompletely defined. Here we show that the short-chain fatty acid crotonate, which is used to study histone crotonylation, changes cell morphology and immune interactions of
. Crotonate reduces invasive hyphal morphogenesis of
within macrophages, thereby delaying macrophage killing and pathogen escape, as well as reducing inflammatory cytokine maturation. Crotonate's ability to reduce hyphal growth is environmentally contingent and pronounced within macrophages. Moreover, crotonate is a stronger hyphal inhibitor than butyrate under the conditions that we tested. Crotonate causes increased histone crotonylation in
under hyphal growth conditions and reduces transcription of hyphae-induced genes in a manner that involves the Nrg1 repressor pathway. Increasing histone acetylation by histone deacetylase inhibition partially rescues hyphal growth and gene transcription in the presence of crotonate. These results indicate that histone crotonylation might compete with acetylation in the regulation of hyphal morphogenesis. Based on our findings, we propose that diverse acylations of histones (and likely also non-histone proteins) enable
to respond to environmental signals, which in turn regulate its cell morphology and host-pathogen interactions.IMPORTANCEMacrophages curtail the proliferation of the pathogen
within human body niches. Within macrophages,
adapts its metabolism and switches to invasive hyphal morphology. These adaptations enable fungal growth and immune escape by triggering macrophage lysis. Transcriptional programs regulate these metabolic and morphogenetic adaptations. Here we studied the roles of chromatin in these processes and implicate lysine crotonylation, a histone mark regulated by metabolism, in hyphal morphogenesis and macrophage interactions by
. We show that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of
. Crotonate represses hyphal gene expression, and we propose that
uses diverse acylation marks to regulate its cell morphology in host environments. Hyphal formation is a virulence property of
. Therefore, a further importance of our study stems from identifying crotonate as a hyphal inhibitor.
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