Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA) reducing its translation ...and stability. They also commonly induce shortening of the mRNA 3' poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3' UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.
Alternative polyadenylation (APA) represents an important mechanism for regulating isoform-specific translation efficiency, stability, and localisation. Though some progress has been made in ...understanding its consequences in metazoans, the role of APA in the model organism Saccharomyces cerevisiae remains a relative mystery because, despite abundant studies on the translational state of mRNA, none differentiate mRNA isoforms’ alternative 3′-end. This review discusses the implications of alternative polyadenylation in S. cerevisiae using other organisms to draw inferences. Given the foundational role that research in this yeast has played in the discovery of the mechanisms of cleavage and polyadenylation and in the drivers of APA, it is surprising that such an inference is required. However, because advances in ribosome profiling are insensitive to APA, how it impacts translation is still unclear. To bridge the gap between widespread observed APA and the discovery of any functional consequence, we also provide a review of the experimental techniques used to uncover the functional importance of 3′ UTR isoforms on translation.
Fungal infections are a global threat, but treatments are limited due to a paucity in antifungal drug targets and the emergence of drug-resistant fungi such as Candida auris. Metabolic adaptations ...enable microbial growth in nutrient-scarce host niches, and they further control immune responses to pathogens, thereby offering opportunities for therapeutic targeting. Because it is a relatively new pathogen, little is known about the metabolic requirements for C. auris growth and its adaptations to counter host defenses. Here, we establish that triggering metabolic dysfunction is a promising strategy against C. auris. Treatment with pyrvinium pamoate (PP) induced metabolic reprogramming and mitochondrial dysfunction evident in disrupted mitochondrial morphology and reduced tricarboxylic acid (TCA) cycle enzyme activity. PP also induced changes consistent with disrupted iron homeostasis. Nutrient supplementation experiments support the proposition that PP-induced metabolic dysfunction is driven by disrupted iron homeostasis, which compromises carbon and lipid metabolism and mitochondria. PP inhibited C. auris replication in macrophages, which is a relevant host niche for this yeast pathogen. We propose that PP causes a multipronged metabolic hit to C. auris: it restricts the micronutrient iron to potentiate nutritional immunity imposed by immune cells, and it further causes metabolic dysfunction that compromises the utilization of macronutrients, thereby curbing the metabolic plasticity needed for growth in host environments. Our study offers a new avenue for therapeutic development against drug-resistant C. auris, shows how complex metabolic dysfunction can be caused by a single compound triggering antifungal inhibition, and provides insights into the metabolic needs of C. auris in immune cell environments.
Over the last decade, Candida auris has emerged as a human pathogen around the world causing life-threatening infections with wide-spread antifungal drug resistance, including pandrug resistance in some cases. In this study, we addressed the mechanism of action of the antiparasitic drug pyrvinium pamoate against C. auris and show how metabolism could be inhibited to curb C. auris proliferation. We show that pyrvinium pamoate triggers sweeping metabolic and mitochondrial changes and disrupts iron homeostasis. PP-induced metabolic dysfunction compromises the utilization of both micro- and macronutrients by C. auris and reduces its growth
and in immune phagocytes. Our findings provide insights into the metabolic requirements for C. auris growth and define the mechanisms of action of pyrvinium pamoate against C. auris, demonstrating how this compound works by inhibiting the metabolic flexibility of the pathogen. As such, our study characterizes credible avenues for new antifungal approaches against C. auris.
Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic ...intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous
and
characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g.
,
,
), metastatic virulence (e.g.
,
), metastatic suppression (e.g.
,
,
,
,
,
) or metastatic avirulence (e.g.
). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC.This article has an associated First Person interview with the first author of the paper.
Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3’ end formation ...factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an
rpb1
mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.
The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning ...transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. ...We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.
Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin ...A1 is an abundant multi-functional Ca
binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24
/Sca1
population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.
We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p. We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging ...of a number of cargo molecules is disrupted when mutations are introduced at this site. Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree. We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p. Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p. Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.
The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH ...zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3′ UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.
•Endo-mesoderm fate is a product of both SKN-1 presence and NEG-1 absence•POS-1 represses expression of neg-1 by preventing cytoplasmic polyadenylation•Transcriptome-wide quantification shows that poly(A) tail length changes in C. elegans•GLD-3/Bicaudal-C function is conserved in embryonic fate specification
The RNA-binding protein POS-1 promotes endo-mesoderm development in C. elegans embryos. Elewa et al. show that POS-1 functions by opposing the cytoplasmic polyadenylation of the gut antagonist neg-1, therefore preventing neg-1 accumulation and allowing endo-mesoderm development. Transcriptome-wide studies suggest a similar regulation of other embryonic transcripts.
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