An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. ...Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode
, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined messenger RNAs (mRNAs) associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1 Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 which encode the RNA regulators SPN-4 and MEG-1 We found that LIN-41 represses translation of
and
, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.
Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into ...the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions.
The yeast Candida albicans colonizes several sites in the human body and responds to metabolic signals in commensal and pathogenic states. The yeast-to-hyphae transition correlates with virulence, ...but how metabolic status is integrated with this transition is incompletely understood. We used the putative mitochondrial fission inhibitor mdivi-1 to probe the crosstalk between hyphal signaling and metabolism. Mdivi-1 repressed C. albicans hyphal morphogenesis, but the mechanism was independent of its presumed target, the mitochondrial fission GTPase Dnm1. Instead, mdivi-1 triggered extensive metabolic reprogramming, consistent with metabolic stress, and reduced endogenous nitric oxide (NO) levels. Limiting endogenous NO stabilized the transcriptional repressor Nrg1 and inhibited the yeast-to-hyphae transition. We establish a role for endogenous NO signaling in C. albicans hyphal morphogenesis and suggest that NO regulates a metabolic checkpoint for hyphal growth. Furthermore, identifying NO signaling as an mdivi-1 target could inform its therapeutic applications in human diseases.
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•The mitochondrial fission inhibitor mdivi-1 represses Candida hyphal morphogenesis•Inhibition of hyphal growth by mdivi-1 is independent of its putative target, Dnm1•Mdivi-1 reprograms metabolic gene expression and reduces endogenous NO levels•Endogenous NO signaling controls hyphal morphogenesis in Candida
Hyphal morphogenesis contributes to virulence of the human fungal pathogen Candida albicans. Koch et al. show that mdivi-1, a putative inhibitor of mitochondrial division, represses hyphal growth of Candida and implicate regulation of endogenous nitric oxide levels in the mechanism of action of mdivi-1 and the regulation of hyphal morphogenesis.
The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the ...poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.
Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For ...most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression.
The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic ...processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.
FGF13 promotes metastasis of triple‐negative breast cancer Johnstone, Cameron N.; Pattison, Andrew D.; Harrison, Paul F. ...
International journal of cancer,
1 July 2020, 2020-07-01, 2020-07-00, 20200701, Volume:
147, Issue:
1
Journal Article
Peer reviewed
Open access
Triple‐negative breast cancer (TNBC) represents 10–20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant ...metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA‐MB‐231 human triple‐negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA‐MB‐231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA‐MB‐231_HM). Gene expression profiling of primary tumours by RNA‐Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA‐MB‐231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal‐like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA‐MB‐231_HM TNBC cells but not in oestrogen receptor (ER)‐positive MCF‐7 or MDA‐MB‐361 cells. However, despite augmenting MDA‐MB‐231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA‐MB‐231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.
What's new?
Triple‐negative breast cancer (TNBC) has a high degree of molecular diversity, with multiple subtypes and varying metastatic behavior. To better understand TNBC heterogeneity, the authors of this study performed RNA expression analysis on four isogenic MDA‐MB‐231 human TNBC tumour models characterized by distinct metastatic capacities. The three metastatic variants upregulation of the fibroblast growth factor FGF13. Elevated FGF13 expression was associated with poor survival in patients with basal‐like breast cancer. In mice, FGF13 knockdown in highly metastatic MDA‐MB‐231 cells reduced spontaneous metastasis to liver and lung without affecting primary xenograft growth, suggesting that FGF13 is a promising antimetastatic target.
Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene ...expression to that mediated by the promoter and coding regions because native 3' UTR are replaced. Here we show that the 3' UTRs, CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3' -end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3' -truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3' -end formation by alternative polyadenylation and antisense transcription from a universal 3' UTR. This means that these 3' UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3' -end formation and regulatory non-coding transcription.
Signal transducer and activator of transcription 3 (STAT3) is a potent transcription factor necessary for life whose activity is corrupted in diverse diseases, including cancer. STAT3 biology was ...presumed to be entirely dependent on its activity as a transcription factor until the discovery of a mitochondrial pool of STAT3, which is necessary for normal tissue function and tumorigenesis. However, the mechanism of this mitochondrial activity remained elusive. This study uses immunoprecipitation and mass spectrometry to identify a complex containing STAT3, leucine-rich pentatricopeptide repeat containing (LRPPRC), and SRA stem-loop-interacting RNA-binding protein (SLIRP) that is required for the stability of mature mitochondrially encoded mRNAs and transport to the mitochondrial ribosome. Moreover, we show that this complex is enriched in patients with lung adenocarcinoma and that its deletion inhibits the growth of lung cancer in vivo, providing therapeutic opportunities through the specific targeting of the mitochondrial activity of STAT3.
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•Unbiased MS shows that mitochondrial STAT3 binds to the LRPPRC-SLIRP complex•STAT3 regulated mitochondrial mRNA stability, ribosomal delivery, and translation kinetics•The STAT3-LRPPRC-SLIRP complex is enriched in LUAD patient tissue•Blocking STAT3 binding to LRPPRC reduces tumor formation in vivo
Fernando et al. describe a STAT3-LRPPRC-SLIRP complex critical for the regulation of mitochondrial mRNA stability and translation kinetics. They show increased abundance of this complex in LUAD compared to adjacent healthy tissue, and the loss of this complex impedes tumor growth in vivo.
The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs ...encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor
mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding,
mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor
and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.
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