RESUMEN
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.
Asunto(s)
Antifúngicos , Complejo de la Endopetidasa Proteasomal , Humanos , Antifúngicos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis , Fluconazol , Candida albicans/metabolismo , Tolerancia a Medicamentos , Ergosterol , Farmacorresistencia Fúngica , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Poliadenilación , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular/genéticaRESUMEN
Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-ß-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.
Asunto(s)
Regulación de la Expresión Génica , Poliadenilación , Humanos , Transición Epitelial-Mesenquimal/genética , Secuencia de Bases , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3'RESUMEN
Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' untranslated regions (3'-UTRs), we observed shifts from promoter proximal regions to the 3'-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Poliadenilación/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Regiones no Traducidas 3'/genética , Línea Celular Tumoral , Humanos , Células MCF-7 , Poli A/genética , Unión Proteica/genética , ARN Polimerasa II/genética , Precursores del ARN/genética , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 5'/genética , Codón Iniciador/metabolismo , Codón de Terminación/metabolismo , Movimiento , Nucleótidos/genética , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Caperuzas de ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Ribosomas/químicaRESUMEN
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.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Subunidades de Proteína/metabolismo , Línea Celular , Respiración de la Célula , Supervivencia Celular/genética , Complejo I de Transporte de Electrón/genética , Edición Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Moleculares , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , ProteómicaRESUMEN
Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.
Asunto(s)
Regiones no Traducidas 3' , Biomarcadores de Tumor/genética , Empalme Alternativo , Biomarcadores de Tumor/metabolismo , Biología Computacional , Bases de Datos de Ácidos Nucleicos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Poliadenilación , Sitios de Empalme de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq , Análisis de la Célula IndividualRESUMEN
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.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/genética , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Células Madre Neoplásicas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transcriptoma , Neoplasias de la Mama Triple Negativas/genética , Regulación hacia ArribaRESUMEN
Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) ß stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Salmonella Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation.
Asunto(s)
Interferón Tipo I/genética , MicroARNs/genética , Isoformas de ARN/genética , Salmonella typhimurium/fisiología , Biología Computacional , Células Dendríticas , Fibroblastos , Perfilación de la Expresión Génica , Humanos , Procesamiento de Término de ARN 3' , Interferencia de ARN , Infecciones por Salmonella/microbiología , Análisis de Secuencia de ARNRESUMEN
The noncoding elements of an mRNA influence multiple aspects of its fate. For example, 3'-UTRs serve as physical and sequence-based information hubs that direct the time, place, and level of translation of the protein encoded in cis, but often also have additional roles in trans. Understanding the information content of 3'-UTRs has been a challenge. Bioinformatic searches for motifs, such as those that encode the polyadenylation signal or microRNA seed regions, are simple enough, but rarely do these inferred positions in genomes correlate well with the actual sites chosen by the relevant nanomachines in living cells. This is almost certainly due to three-dimensional complexity of RNA, the physical states of which are recognized by RNA-binding proteins that serve to read and interpret the information content. Here, we follow the 3'-UTR-mediated posttranscriptional metabolism of mRNA in the germline of the nematode worm Caenorhabditis elegans. While many areas still require the clarification only detailed fundamental research can provide, this model system can serve as a basis of 3'-mediated regulatory control for elaboration in more complex metazoan systems.
Asunto(s)
Regiones no Traducidas 3' , Regulación de la Expresión Génica , ARN Mensajero , Regiones no Traducidas 3'/genética , Secuencias de Aminoácidos , Animales , Caenorhabditis elegans , Poliadenilación , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , TiempoRESUMEN
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.
Asunto(s)
Regiones no Traducidas 3' , Regiones Promotoras Genéticas , ARN sin Sentido , Transcripción Genética , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Citocromos c/genética , Regulación Fúngica de la Expresión Génica , Sitios Genéticos , Poliadenilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Levaduras/genéticaRESUMEN
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.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/genética , Proteínas Fúngicas/genética , Redes Reguladoras de Genes , Proteínas de Unión al ARN/biosíntesis , Proteínas de Saccharomyces cerevisiae/biosíntesis , Adaptación Fisiológica/genética , Candida albicans/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Ribonucleasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN/métodos , Regiones no Traducidas 3' , Regulación Fúngica de la Expresión Génica , Estabilidad del ARN , ARN de Hongos/análisis , Ribonucleasas/deficiencia , Ribonucleasas/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , TranscriptomaRESUMEN
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.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Intrones/genética , Proteína II de Unión a Poli(A)/genética , Estabilidad del ARN/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cryptococcus neoformans/genética , Redes y Vías Metabólicas/genética , Poli A/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/fisiología , Poro Nuclear/metabolismo , Proteínas Represoras/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Factor 4E Eucariótico de Iniciación/genética , Mutación/genética , Oogénesis/genética , Biosíntesis de Proteínas , Transporte de Proteínas , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Procesos de Determinación del SexoRESUMEN
The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Sitios de Unión , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Conformación de Ácido Nucleico , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Unión Proteica , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The controlled biogenesis of mitochondria is a key cellular system coordinated with the cell division cycle, and major efforts in systems biology currently are directed toward understanding of the control points at which this coordination is achieved. Here we present insights into the function, evolution, and regulation of mitochondrial biogenesis through the study of the protein import machinery in the human fungal pathogen, Candida albicans. Features that distinguish C. albicans from baker's yeast (Saccharomyces cerevisiae) include the stringency of metabolic control at the level of oxygen consumption, the potential for ATP exchange through the porin in the outer membrane, and components and domains in the sorting and assembling machinery complex, a molecular machine that drives the assembly of proteins in the outer mitochondrial membrane. Analysis of targeting sequences and assays of mitochondrial protein import show that components of the electron transport chain are imported by distinct pathways in C. albicans and S. cerevisiae, representing an evolutionary rewiring of mitochondrial import pathways. We suggest that studies using this pathogen as a model system for mitochondrial biogenesis will greatly enhance our knowledge of how mitochondria are made and controlled through the course of the cell-division cycle.
Asunto(s)
Evolución Biológica , Candida albicans/fisiología , Proteínas Portadoras/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Análisis por Conglomerados , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Cadenas de Markov , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Consumo de Oxígeno/fisiología , Filogenia , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Complejo Mediador , Saccharomyces cerevisiae , Biopelículas/crecimiento & desarrollo , Candida albicans/patogenicidad , Proteínas Fúngicas/metabolismo , Regulación de la Expresión Génica/genética , Complejo Mediador/genética , Complejo Mediador/metabolismo , Estructura Terciaria de Proteína/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Especificidad de la Especie , Virulencia/genéticaRESUMEN
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.
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ARN Mensajero/análisis , ARN/química , Animales , ADN Polimerasa I/química , Técnicas Genéticas , Oligodesoxirribonucleótidos/análisis , Reacción en Cadena de la PolimerasaRESUMEN
Reporter-based studies support inhibition of translation at the level of initiation as a substantial component of the miRNA mechanism, yet recent global analyses have suggested that they predominantly act through decreasing target mRNA stability. Cells commonly coexpress several processing isoforms of an mRNA, which may also differ in their regulatory untranslated regions (UTR). In particular, cancer cells are known to express high levels of short 3' UTR isoforms that evade miRNA-mediated regulation, whereas longer 3' UTRs predominate in nontransformed cells. To test whether mRNA isoform diversity can obscure detection of miRNA-mediated control at the level of translation, we assayed the responses of 11 endogenous let-7 targets to inactivation of this miRNA in HeLa cells, an intensively studied model system. We show that translational regulation in many cases appears to be modest when measuring the composite polysome profile of all extant isoforms of a given mRNA by density ultracentrifugation. In contrast, we saw clear effects at the level of translation initiation for multiple examples when selectively profiling mRNA isoforms carrying the 5' or 3' untranslated regions that were actually permissive to let-7 action, or when let-7 and a second targeting miRNA were jointly manipulated. Altogether, these results highlight a caveat to the mechanistic interpretation of data from global miRNA target analyses in transformed cells. Importantly, they reaffirm the importance of translational control as part of the miRNA mechanism in animal cells.