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1.
RNA ; 26(8): 969-981, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32295865

RESUMO

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.


Assuntos
Neoplasias da Mama/genética , Poliadenilação/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Poli A/genética , Ligação Proteica/genética , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Mensageiro/genética
2.
Nucleic Acids Res ; 44(1): 377-86, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26481348

RESUMO

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.


Assuntos
Regiões 3' não Traduzidas , Regiões Promotoras Genéticas , RNA Antissenso , Transcrição Gênica , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Citocromos c/genética , Regulação Fúngica da Expressão Gênica , Loci Gênicos , Poliadenilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/genética
3.
Elife ; 102021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232857

RESUMO

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.


Assuntos
Poli A/química , Poliadenilação , Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas , DNA Helicases , Cinética , RNA Helicases , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae
4.
Cell Rep ; 31(3): 107528, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32320659

RESUMO

Identification of multiple histone acylations diversifies transcriptional control by metabolism, but their functions are incompletely defined. Here we report evidence of histone crotonylation in the human fungal pathogen Candida albicans. We define the enzymes that regulate crotonylation and show its dynamic control by environmental signals: carbon sources, the short-chain fatty acids butyrate and crotonate, and cell wall stress. Crotonate regulates stress-responsive transcription and rescues C. albicans from cell wall stress, indicating broad impact on cell biology. The YEATS domain crotonylation readers Taf14 and Yaf9 are required for C. albicans virulence, and Taf14 controls gene expression, stress resistance, and invasive growth via its chromatin reader function. Blocking the Taf14 C terminus with a tag reduced virulence, suggesting that inhibiting Taf14 interactions with chromatin regulators impairs function. Our findings shed light on the regulation of histone crotonylation and the functions of the YEATS proteins in eukaryotic pathogen biology and fungal infections.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Animais , Candida albicans/patogenicidade , Cromatina/metabolismo , Crotonatos/metabolismo , Feminino , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Domínios Proteicos , Fator de Transcrição TFIID , Virulência
5.
Methods Mol Biol ; 2049: 141-164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31602610

RESUMO

Next-generation sequencing (NGS) and its application to RNA (RNA-seq) has opened up multiple aspects of RNA processing to deep transcriptome-wide analysis at nucleotide resolution. This has been useful in delineating the transcribed areas of the genome, and in quantitation of RNA isoforms. Such isoforms can diversify the regulatory repertoire of mRNAs. For example, the 3'-end of mRNA can vary in two important ways, in the position chosen for cleavage and polyadenylation, and in the length of the poly(A)-tail. Accordingly, the step-up in resolution made possible by NGS has revealed an unexpectedly high level of alternative polyadenylation (APA). Moreover, it has massively simplified the transcriptome-wide detection of poly(A)-tail length changes. Here we present our approach to the study of 3'-end dynamics using a 3'-focused RNA-seq method called PAT-seq (for poly(A)-test sequencing). The approach records gene expression, APA, and poly(A)-tail changes between transcriptomes to reveal complex interplay between transcriptional and posttranscriptional control mechanisms.


Assuntos
Poli A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Poli A/genética , Poliadenilação , Análise de Sequência de RNA
6.
PLoS One ; 7(9): e46243, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23029448

RESUMO

Understanding multicellular fungal structures is important for designing better strategies against human fungal pathogens. For example, the ability to form multicellular biofilms is a key virulence property of the yeast Candida albicans. C. albicans biofilms form on indwelling medical devices and are drug resistant, causing serious infections in hospital settings. Multicellular fungal communities are heterogeneous, consisting of cells experiencing different environments. Heterogeneity is likely important for the phenotypic characteristics of communities, yet it is poorly understood. Here we used colonies of the yeast Saccharomyces cerevisiae as a model fungal multicellular structure. We fractionated the outside colony layers from the cells in the center by FACS, using a Cit1-GFP marker expressed exclusively on the outside. Transcriptomics analysis of the two subpopulations revealed that the outside colony layers are actively growing by fermentative metabolism, while the cells residing on the inside are in a resting state and experience changes to mitochondrial activity. Our data shows several parallels with C. albicans biofilms providing insight into the contributions of heterogeneity to biofilm phenotypes. Hallmarks of C. albicans biofilms - the expression of ribosome and translation functions and activation of glycolysis and ergosterol biosynthesis occur on the outside of colonies, while expression of genes associates with sulfur assimilation is observed in the colony center. Cell wall restructuring occurs in biofilms, and cell wall functions are enriched in both fractions: the outside cells display enrichment of cell wall biosynthesis enzymes and cell wall proteins, while the inside cells express cell wall degrading enzymes. Our study also suggests that noncoding transcription and posttranscriptional mRNA regulation play important roles during growth of yeast in colonies, setting the scene for investigating these pathways in the development of multicellular fungal communities.


Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Heterogeneidade Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcriptoma , Candida albicans/genética , Candida albicans/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Redes e Vias Metabólicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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