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1.
RNA ; 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844344

RESUMO

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue specific and essential for some tissue development, such as liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by employing a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays more relevant role in small RNA metabolism and ribosome biogenesis.

2.
Nucleic Acids Res ; 51(22): 12076-12091, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37950888

RESUMO

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.


Assuntos
Desenvolvimento Embrionário , Oócitos , Biossíntese de Proteínas , Regulação da Expressão Gênica no Desenvolvimento , Meiose , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Camundongos
3.
PLoS Pathog ; 14(10): e1007377, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30346988

RESUMO

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.


Assuntos
Evolução Molecular , Proteínas Fúngicas/genética , Kluyveromyces/genética , Regiões Promotoras Genéticas , Elementos de Resposta , Transcrição Gênica , Vírus/genética , Sequência de Bases , Citoplasma , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Poliadenilação , Estabilidade de RNA , Homologia de Sequência
4.
Cytokine ; 133: 155141, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32615410

RESUMO

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.


Assuntos
Anexina A2/metabolismo , Membrana Celular/metabolismo , Interleucina-1alfa/metabolismo , Estresse Oxidativo/fisiologia , Actinas/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Humanos , Fosforilação/fisiologia , Proteínas S100/metabolismo
5.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070012

RESUMO

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.


Assuntos
Membrana Nuclear/genética , Oócitos/crescimento & desenvolvimento , Polirribossomos/genética , Proteínas de Ligação a RNA/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Meiose/genética , Camundongos , Membrana Nuclear/metabolismo , Oócitos/metabolismo , RNA Mensageiro Estocado/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética , RNA-Seq
6.
Mol Genet Genomics ; 293(1): 167-186, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28942592

RESUMO

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.


Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Proteínas de Ligação ao Cap de RNA/genética , Splicing de RNA/genética , Regiões 3' não Traduzidas , Encéfalo/metabolismo , Linhagem Celular , Fator de Iniciação 4E em Eucariotos/metabolismo , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica , Humanos , Placenta/metabolismo , Poliadenilação/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Int J Mol Sci ; 19(9)2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30235877

RESUMO

The rate of chromosome segregation errors that emerge during meiosis I in the mammalian female germ line are known to increase with maternal age; however, little is known about the underlying molecular mechanism. The objective of this study was to analyze meiotic progression of mouse oocytes in relation to maternal age. Using the mouse as a model system, we analyzed the timing of nuclear envelope breakdown and the morphology of the nuclear lamina of oocytes obtained from young (2 months old) and aged females (12 months old). Oocytes obtained from older females display a significantly faster progression through meiosis I compared to the ones obtained from younger females. Furthermore, in oocytes from aged females, lamin A/C structures exhibit rapid phosphorylation and dissociation. Additionally, we also found an increased abundance of MPF components and increased translation of factors controlling translational activity in the oocytes of aged females. In conclusion, the elevated MPF activity observed in aged female oocytes affects precocious meiotic processes that can multifactorially contribute to chromosomal errors in meiosis I.


Assuntos
Envelhecimento/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose , Oócitos/metabolismo , Envelhecimento/genética , Animais , Feminino , Fator Promotor de Maturação/genética , Mesotelina , Camundongos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Oócitos/citologia , Fosforilação , Processamento de Proteína Pós-Traducional
8.
Croat Med J ; 58(3): 203-213, 2017 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-28613037

RESUMO

AIM: A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. METHODS: Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. RESULTS: Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. CONCLUSION: The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.


Assuntos
Osso e Ossos/química , DNA/análise , República Tcheca , Impressões Digitais de DNA/normas , Genética Forense , Humanos
9.
BMC Mol Biol ; 17(1): 21, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27578149

RESUMO

BACKGROUND: Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. RESULTS: We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. CONCLUSION: Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Resposta ao Choque Térmico , Estresse Oxidativo , Proteínas de Ligação ao Cap de RNA/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Citosol/metabolismo , Fator de Iniciação 4E em Eucariotos/análise , Fator de Iniciação 4E em Eucariotos/genética , Células HEK293 , Humanos , Proteína I de Ligação a Poli(A)/análise , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação ao Cap de RNA/análise , Proteínas de Ligação ao Cap de RNA/genética , RNA Mensageiro/genética , Alinhamento de Sequência
10.
BMC Microbiol ; 16(1): 187, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27527702

RESUMO

BACKGROUND: Sequence variability in the hepatitis C virus (HCV) genome has led to the development and classification of six genotypes and a number of subtypes. The HCV 5' untranslated region mainly comprises an internal ribosomal entry site (IRES) responsible for cap-independent synthesis of the viral polyprotein and is conserved among all HCV genotypes. DESCRIPTION: Considering the possible high impact of variations in HCV IRES on viral protein production and thus virus replication, we decided to collect the available data on known nucleotide variants in the HCV IRES and their impact on IRES function in translation initiation. The HCV IRES variation database (HCVIVdb) is a collection of naturally occurring and engineered mutation entries for the HCV IRES. Each entry contains contextual information pertaining to the entry such as the HCV genotypic background and links to the original publication. Where available, quantitative data on the IRES efficiency in translation have been collated along with details on the reporter system used to generate the data. Data are displayed both in a tabular and graphical formats and allow direct comparison of results from different experiments. Together the data provide a central resource for researchers in the IRES and hepatitis C-oriented fields. CONCLUSION: The collation of over 1900 mutations enables systematic analysis of the HCV IRES. The database is mainly dedicated to detailed comparative and functional analysis of all the HCV IRES domains, which can further lead to the development of site-specific drug designs and provide a guide for future experiments. HCVIVdb is available at http://www.hcvivdb.org .


Assuntos
Bases de Dados Genéticas , Hepacivirus/genética , Sítios Internos de Entrada Ribossomal/genética , Regiões 5' não Traduzidas , Sequência de Bases , Coleta de Dados , Genótipo , Hepacivirus/metabolismo , Hepatite C/virologia , Humanos , Mutação , Biossíntese de Proteínas , RNA Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
11.
Mol Ecol Resour ; 23(8): 1800-1811, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37561110

RESUMO

Metagenomics provides a tool to assess the functional potential of environmental and host-associated microbiomes based on the analysis of environmental DNA: assembly, gene prediction and annotation. While gene prediction is straightforward for most bacterial and archaeal taxa, it has limited applicability in the majority of eukaryotic organisms, including fungi that contain introns in gene coding sequences. As a consequence, eukaryotic genes are underrepresented in metagenomics datasets and our understanding of the contribution of fungi and other eukaryotes to microbiome functioning is limited. Here, we developed a machine intelligence-based algorithm that predicts fungal introns in environmental DNA with reasonable precision and used it to improve the annotation of environmental metagenomes. Intron removal increased the number of predicted genes by up to 9.1% and improved the annotation of several others. The proportion of newly predicted genes increased with the share of eukaryotic genes in the metagenome and-within fungal taxa-increased with the number of introns per gene. Our approach provides a tool named SVMmycointron for improved metagenome annotation, especially of microbiomes with a high proportion of eukaryotes. The scripts described in the paper are made publicly available and can be readily utilized by microbiome researchers analysing metagenomics data.

12.
Nucleic Acids Res ; 38(Database issue): D131-6, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19917642

RESUMO

The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The supplementary material contains an updated list of reported IRESs.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , RNA Viral/genética , Ribossomos/genética , Animais , Biologia Computacional/tendências , Genoma Viral , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/genética , Plasmídeos/metabolismo , Análise de Sequência de DNA , Software
13.
Proc Natl Acad Sci U S A ; 106(6): 1954-9, 2009 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-19193860

RESUMO

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.


Assuntos
Perfilação da Expressão Gênica , Genoma Fúngico , Lignina/metabolismo , Redes e Vias Metabólicas/genética , Polyporales/genética , Sequência de Bases , Evolução Biológica , Celulases , Enzimas/genética , Glicosídeo Hidrolases , Dados de Sequência Molecular , Oxirredutases , Polyporales/metabolismo , Madeira/metabolismo
14.
Eur J Cell Biol ; 101(2): 151210, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35240557

RESUMO

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation. In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.


Assuntos
Proteínas Imediatamente Precoces , Fator Promotor de Maturação , Animais , Pontos de Checagem do Ciclo Celular , Feminino , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Mamíferos/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose , Prófase Meiótica I , Camundongos , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética
15.
Forensic Sci Int Genet ; 54: 102538, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34265517

RESUMO

Research on ancient and forensic DNA is related in many ways, and the two fields must deal with similar obstacles. Therefore, communication between these two communities has the potential to improve results in both research fields. Here, we present the insights gained in the ancient DNA community with regard to analyzing DNA from aged skeletal material and the potential use of the developed protocols in forensic work. We discuss the various steps, from choosing samples for DNA extraction to deciding between classical PCR amplification and massively parallel sequencing approaches. Based on the progress made in ancient DNA analyses combined with the requirements of forensic work, we suggest that there is substantial potential for incorporating ancient DNA approaches into forensic protocols, a process that has already begun to a considerable extent. However, taking full advantage of the experiences gained from ancient DNA work will require comparative studies by the forensic DNA community to tailor the methods developed for ancient samples to the specific needs of forensic studies and case work. If successful, in our view, the benefits for both communities would be considerable.


Assuntos
DNA Antigo , DNA , Idoso , DNA/genética , Degradação Necrótica do DNA , Impressões Digitais de DNA , Genética Forense , Humanos
16.
Commun Biol ; 4(1): 788, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172827

RESUMO

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.


Assuntos
Blastocisto/fisiologia , Endoderma/citologia , Biossíntese de Proteínas , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Embrionário , Camundongos , Proteínas de Ligação a RNA/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
17.
RNA ; 14(9): 1720-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18697919

RESUMO

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.


Assuntos
Regulação da Expressão Gênica , Luciferases de Vaga-Lume/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Linhagem Celular , DNA Complementar/genética , Hepacivirus/genética , Humanos
18.
FEMS Yeast Res ; 10(5): 558-69, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20491942

RESUMO

The eukaryotic translation initiation factor 4E is an essential and highly conserved protein. As a part of the translational machinery, it plays a key role in the recruitment of mRNA via binding to its m(7)GpppN 5' terminal cap structure. The opportunistic human pathogen Candida albicans is the only known eukaryotic organism with the ability to survive defects in mRNA capping, which suggests unique features of its eIF4E protein. Here, we provide the first experimental evidence of the function of the C. albicans putative gene orf19.7626 as an eIF4E protein. We also show that Ca4E(Leu116) and Ca4E(Ser116) protein variants, both of which occur naturally in C. albicans due to the ambiguous decoding of the CUG(116) codon, display different sensitivities to elevated temperature. Our results clearly point to the importance of the S4-H4 loop for the function of the Ca4E translation initiation factor, and suggest the possible regulatory role of the codon-reading ambiguity within this loop in C. albicans. We proved Saccharomyces cerevisiae as a useful tool organism for studies of C. albicans translation initiation apparatus.


Assuntos
Candida albicans/genética , Códon , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas Fúngicas/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/efeitos da radiação , Genes Essenciais , Genes Fúngicos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência , Temperatura
19.
Wiley Interdiscip Rev RNA ; 11(6): e1596, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32362075

RESUMO

Stress granules (SGs), hallmarks of the cellular adaptation to stress, promote survival, conserve cellular energy, and are fully dissolved upon the cessation of stress treatment. Different stresses can initiate the assembly of SGs, but arsenite and heat are the best studied of these stresses. The composition of SGs and posttranslational modifications of SG proteins differ depending on the type and severity of the stress insult, methodology used, cell line, and presence of overexpressed and tagged proteins. A group of 18 proteins showing differential localization to SGs in heat- and arsenite-stressed mammalian cell lines is described. Upon severe and prolonged stress, physiological SGs transform into more solid protein aggregates that are no longer reversible and do not contain mRNA. Similar pathological inclusions are hallmarks of neurodegenerative diseases. SGs induced by heat stress are less dynamic than SGs induced by arsenite and contain a set of unique proteins and linkage-specific polyubiquitinated proteins. The same types of ubiquitin linkages have been found to contribute to the development of neurodegenerative disorders such as Parkinson disease, Alzheimer disease, and amyotrophic lateral sclerosis (ALS). We propose heat stress-induced SGs as a possible model of an intermediate stage along the transition from dynamic, fully reversible arsenite stress-induced SGs toward aberrant SGs, the hallmark of neurodegenerative diseases. Stress- and methodology-specific differences in the compositions of SGs and the transition of SGs to aberrant protein aggregates are discussed. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization.


Assuntos
Arsenitos/metabolismo , Grânulos Citoplasmáticos/metabolismo , Temperatura Alta , Animais , Humanos , Processamento de Proteína Pós-Traducional , Estresse Fisiológico
20.
Aging Cell ; 19(10): e13231, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32951297

RESUMO

Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age-related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.


Assuntos
Oócitos/metabolismo , Fatores Etários , Animais , Feminino , Humanos , Mamíferos
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