Publications by authors named "Martin Pospísek"

28 Publications

  • Page 1 of 1

Age-related differences in the translational landscape of mammalian oocytes.

Aging Cell 2020 10 20;19(10):e13231. Epub 2020 Sep 20.

Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal Physiology and Genetics, CAS, Libechov, Czech Republic.

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.
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http://dx.doi.org/10.1111/acel.13231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576272PMC
October 2020

Co-localization of Interleukin-1α and Annexin A2 at the plasma membrane in response to oxidative stress.

Cytokine 2020 09 30;133:155141. Epub 2020 Jun 30.

Department of Biomedicine, Faculty of Medicine, University of Bergen, Bergen, Norway.

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.
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http://dx.doi.org/10.1016/j.cyto.2020.155141DOI Listing
September 2020

Changing faces of stress: Impact of heat and arsenite treatment on the composition of stress granules.

Wiley Interdiscip Rev RNA 2020 11 3;11(6):e1596. Epub 2020 May 3.

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czechia.

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.
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http://dx.doi.org/10.1002/wrna.1596DOI Listing
November 2020

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method.

Int J Mol Sci 2020 Feb 13;21(4). Epub 2020 Feb 13.

Laboratory of RNA Biochemistry, Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 44, 2 Prague, Czech Republic.

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.
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http://dx.doi.org/10.3390/ijms21041254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072993PMC
February 2020

Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5' Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1.

Front Microbiol 2019 30;10:2366. Epub 2019 Oct 30.

Laboratory of RNA Biochemistry, Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czechia.

We employed virus-like elements (VLEs) pGKL1,2 from as a model to investigate the previously neglected transcriptome of the broader group of yeast cytoplasmic linear dsDNA VLEs. We performed 5' and 3' RACE analyses of all pGKL1,2 mRNAs and found them not 3' polyadenylated and containing frequently uncapped 5' poly(A) leaders that are not complementary to VLE genomic DNA. The degree of 5' capping and/or 5' mRNA polyadenylation is specific to each gene and is controlled by the corresponding promoter region. The expression of pGKL1,2 transcripts is independent of eIF4E and Pab1 and is enhanced in Δ and Δ strains. We suggest a model of primitive pGKL1,2 gene expression regulation in which the degree of 5' mRNA capping and 5' non-template polyadenylation, together with the presence of negative regulators such as Pab1 and Lsm1, play important roles. Our data also support a hypothesis of a close relationship between yeast linear VLEs and poxviruses.
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http://dx.doi.org/10.3389/fmicb.2019.02366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831550PMC
October 2019

Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin.

PLoS Pathog 2018 10 22;14(10):e1007377. Epub 2018 Oct 22.

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211774PMC
October 2018

Increased Expression of Maturation Promoting Factor Components Speeds Up Meiosis in Oocytes from Aged Females.

Int J Mol Sci 2018 Sep 19;19(9). Epub 2018 Sep 19.

Laboratory of Biochemistry and Molecular Biology of Germ Cells, Institute of Animal Physiology and Genetics, CAS, Rumburska 89, 277 21 Libechov, Czech Republic.

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.
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http://dx.doi.org/10.3390/ijms19092841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164426PMC
September 2018

Characterization of Hepatitis C Virus IRES Quasispecies - From the Individual to the Pool.

Front Microbiol 2018 24;9:731. Epub 2018 Apr 24.

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czechia.

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus from the genus . The viral genomic +RNA is 9.6 kb long and contains highly structured 5' and 3' untranslated regions (UTRs) and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5' UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES), which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE) analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex population of viral quasispecies better than bare information about their nucleotide sequences. A similar approach might be used for monitoring of sequence variations in quasispecies populations of other RNA viruses in all cases when changes in primary sequence represent changes in measurable and easily quantifiable phenotypes.
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http://dx.doi.org/10.3389/fmicb.2018.00731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928756PMC
April 2018

Major splice variants and multiple polyadenylation site utilization in mRNAs encoding human translation initiation factors eIF4E1 and eIF4E3 regulate the translational regulators?

Mol Genet Genomics 2018 Feb 23;293(1):167-186. Epub 2017 Sep 23.

Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, 128 44, Prague, Czech Republic.

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.
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http://dx.doi.org/10.1007/s00438-017-1375-4DOI Listing
February 2018

Results of a collaborative study on DNA identification of aged bone samples.

Croat Med J 2017 Jun;58(3):203-213

Daniel Vanek, Forensic DNA Service, Budinova 2, 180 81 Prague 8, Czech Republic,

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470125PMC
http://dx.doi.org/10.3325/cmj.2017.58.203DOI Listing
June 2017

Distinct recruitment of human eIF4E isoforms to processing bodies and stress granules.

BMC Mol Biol 2016 08 30;17(1):21. Epub 2016 Aug 30.

Laboratory of RNA Biochemistry, Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, 128 00, Prague 2, Czech Republic.

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.
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http://dx.doi.org/10.1186/s12867-016-0072-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006505PMC
August 2016

HCVIVdb: The hepatitis-C IRES variation database.

BMC Microbiol 2016 08 15;16(1):187. Epub 2016 Aug 15.

Department of Genetics & Microbiology, Faculty of Science, Charles University in Prague, Viničná 5, 128 44, Prague 2, Czech Republic.

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 .
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http://dx.doi.org/10.1186/s12866-016-0804-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986321PMC
August 2016

Decontamination by Persteril 36 may affect the reliability of DNA-based detection of biological warfare agents-short communication.

Folia Microbiol (Praha) 2016 Sep 24;61(5):417-21. Epub 2016 Feb 24.

Forensic DNA Service, s.r.o., Janovskeho 18, Prague 7, 170 00, Czech Republic.

Persteril 36 is a disinfectant with a broad spectrum of antimicrobial activity. Because of its bactericidal, virucidal, fungicidal, and sporicidal effectiveness, it is used as a disinfectant against biological warfare agents in the emergency and army services. In case of an attack with potentially harmful biological agents, a person's gear or afflicted skin is sprayed with a diluted solution of Persteril 36 as a precaution. Subsequently, the remains of the biological agents are analyzed. However, the question remains concerning whether DNA can be successfully analyzed from Persteril 36-treated dead bacterial cells. Spore-forming Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Xanthomonas campestris were splattered on a camouflage suit and treated with 2 or 0.2 % Persteril 36. After the disinfectant vaporized, the bacterial DNA was extracted and quantified by real-time PCR. A sufficient amount of DNA was recovered for downstream analysis only in the case of spore-forming B. subtilis treated with a 0.2 % solution of Persteril 36. The bacterial DNA was almost completely destroyed in Gram-negative bacteria or after treatment with the more concentrated solution in B. subtilis. This phenomenon can lead to false-negative results during the identification of harmful microorganisms.
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http://dx.doi.org/10.1007/s12223-016-0451-1DOI Listing
September 2016

Understanding the potential of hepatitis C virus internal ribosome entry site domains to modulate translation initiation via their structure and function.

Wiley Interdiscip Rev RNA 2015 Mar-Apr;6(2):211-24. Epub 2014 Oct 28.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic.

Translation initiation in the hepatitis C virus (HCV) occurs through a cap-independent mechanism that involves an internal ribosome entry site (IRES) capable of interacting with and utilizing the eukaryotic translational machinery. In this review, we focus on the structural configuration of the different HCV IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. In some cases, multiple mutations, even those scattered across different domains, led to restoration of the translational activity of the HCV IRES, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long-range inter- and/or intra-domain functional interactions. The precise functioning of the HCV IRES requires the specific interaction of its domains with ribosomal subunits and a subset of eukaryotic translation initiation factors (eIFs). The structural conformation, sequence preservation and variability, and translational machinery association with the HCV IRES regions are also thoroughly discussed, along with other factors that can affect and influence the formation of translation initiation complexes.
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http://dx.doi.org/10.1002/wrna.1268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361049PMC
November 2015

Polysome profile analysis--yeast.

Methods Enzymol 2013 ;530:173-81

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, the Czech Republic.

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
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http://dx.doi.org/10.1016/B978-0-12-420037-1.00009-9DOI Listing
March 2014

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

PLoS One 2012 6;7(8):e41801. Epub 2012 Aug 6.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412866PMC
January 2013

Polysome analysis and RNA purification from sucrose gradients.

Methods Mol Biol 2011 ;703:293-309

Department of Genetics and Microbiology, Charles University in Prague, Prague, Czech Republic.

Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.
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http://dx.doi.org/10.1007/978-1-59745-248-9_20DOI Listing
March 2011

Ambiguous decoding of the CUG codon alters the functionality of the Candida albicans translation initiation factor 4E.

FEMS Yeast Res 2010 Aug 7;10(5):558-69. Epub 2010 Apr 7.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

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.
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http://dx.doi.org/10.1111/j.1567-1364.2010.00629.xDOI Listing
August 2010

IRESite--a tool for the examination of viral and cellular internal ribosome entry sites.

Nucleic Acids Res 2010 Jan 16;38(Database issue):D131-6. Epub 2009 Nov 16.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicná 5, 128 44 Prague 2, Czech Republic.

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.
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http://dx.doi.org/10.1093/nar/gkp981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808886PMC
January 2010

Phylogenetic composition and properties of bacteria coexisting with the fungus Hypholoma fasciculare in decaying wood.

ISME J 2009 Oct 11;3(10):1218-21. Epub 2009 Jun 11.

Laboratory of Environmental Microbiology, Institute of Microbiology of the ASCR, v.v.i., Prague 4 14220, Czech Republic.

White-rot fungi are major degraders of woody materials in terrestrial environments because of their ability to decompose lignin. However, little is known on the possible associations of white-rot fungi with other microorganisms during wood decay. We investigated the numbers, community composition and functional traits of bacteria present in natural wood samples under advanced decay by the white-rot basidiomycete Hypholoma fasciculare. The wood samples contained high numbers of cultivable bacteria (0.2-8 x 10(9) colony forming units (CFU) per g of dry wood). Most cultivable bacteria belonged to Proteobacteria and Acidobacteria (75% and 23% of sequences, respectively). The same phyla were also found to be dominant (59% and 23%, respectively) using a non-culturable quantification technique, namely, direct cloning and sequencing of 16sRNA genes extracted from wood. Bacteria that could be subcultured consisted of acid-tolerant strains that seemed to rely on substrates released by lignocellulolytic enzyme activities of the fungus. There were no indications for antagonism (antibiosis) of the bacteria against the fungus.
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http://dx.doi.org/10.1038/ismej.2009.64DOI Listing
October 2009

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.

Proc Natl Acad Sci U S A 2009 Feb 4;106(6):1954-9. Epub 2009 Feb 4.

Los Alamos National Laboratory/Joint Genome Institute, P.O. Box 1663, Los Alamos, NM 87545, USA.

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.
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http://dx.doi.org/10.1073/pnas.0809575106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2644145PMC
February 2009

Firefly luciferase gene contains a cryptic promoter.

RNA 2008 Sep 12;14(9):1720-9. Epub 2008 Aug 12.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.

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.
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http://dx.doi.org/10.1261/rna.831808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525954PMC
September 2008

Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast.

J Gen Virol 2007 Jul;88(Pt 7):1992-2002

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic.

Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic (342)AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.
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http://dx.doi.org/10.1099/vir.0.82782-0DOI Listing
July 2007

IRESite: the database of experimentally verified IRES structures (www.iresite.org).

Nucleic Acids Res 2006 Jan;34(Database issue):D125-30

Charles University, Faculty of Science, Department of Genetics and Microbiology, Vinicna 5, Prague 2, 128 44, Czech Republic.

IRESite is an exhaustive, manually annotated non-redundant relational database focused on the IRES elements (Internal Ribosome Entry Site) and containing information not available in the primary public databases. IRES elements were originally found in eukaryotic viruses hijacking initiation of translation of their host. Later on, they were also discovered in 5'-untranslated regions of some eukaryotic mRNA molecules. Currently, IRESite presents up to 92 biologically relevant aspects of every experiment, e.g. the nature of an IRES element, its functionality/defectivity, origin, size, sequence, structure, its relative position with respect to surrounding protein coding regions, positive/negative controls used in the experiment, the reporter genes used to monitor IRES activity, the measured reporter protein yields/activities, and references to original publications as well as cross-references to other databases, and also comments from submitters and our curators. Furthermore, the site presents the known similarities to rRNA sequences as well as RNA-protein interactions. Special care is given to the annotation of promoter-like regions. The annotated data in IRESite are bound to mostly complete, full-length mRNA, and whenever possible, accompanied by original plasmid vector sequences. New data can be submitted through the publicly available web-based interface at http://www.iresite.org and are curated by a team of lab-experienced biologists.
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http://dx.doi.org/10.1093/nar/gkj081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1347444PMC
January 2006

Rck2 is required for reprogramming of ribosomes during oxidative stress.

Mol Biol Cell 2006 Mar 28;17(3):1472-82. Epub 2005 Dec 28.

Department of Cell and Molecular Biology, Lundberg Laboratory, Göteborg University, S-405 30 Göteborg, Sweden.

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Delta mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Delta cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Delta cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.
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http://dx.doi.org/10.1091/mbc.e05-07-0632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1382333PMC
March 2006

Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.

Int J Parasitol 2005 Jul;35(8):895-910

Department of Parasitology, Faculty of Science, Charles University, Vinicná 7, CZ 12844 Prague, Czech Republic.

Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.
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http://dx.doi.org/10.1016/j.ijpara.2005.02.018DOI Listing
July 2005

Denaturing RNA electrophoresis in TAE agarose gels.

Anal Biochem 2005 Jan;336(1):46-50

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicná 5, 128 44, Prague, Czech Republic.

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.
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http://dx.doi.org/10.1016/j.ab.2004.09.010DOI Listing
January 2005

Intracellular interleukin-1alpha functionally interacts with histone acetyltransferase complexes.

J Biol Chem 2004 Feb 11;279(6):4017-26. Epub 2003 Nov 11.

Department of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, D-89081 Ulm, Germany.

Interleukin-1alpha (IL-1alpha) is an inflammatory cytokine acting extracellularly via membrane receptors. Interestingly, a significant portion of synthesized IL-1alpha is not secreted; instead, it is actively translocated into the cell nucleus. IL-1alpha was indeed shown to be involved in certain intracellular processes, such as control of proliferation, apoptosis, or migration, however, the mechanisms of such actions are not known. Here we show that intracellular IL-1alpha fused to the Gal4p DNA-binding domain (Gal4BD) possesses strong transactivation potential that can be boosted by overexpression of the transcriptional coactivator p300. We demonstrate that the IL-1alpha precursor interacts via its N-terminal peptide (IL-1NTP) with histone acetyltransferases p300, PCAF, Gcn5 and with the adaptor component Ada3, and that it integrates into the PCAF.p300 complex in a non-destructive manner. In analogy with known acidic coactivators, yeast strains expressing Gal4BD/IL-1NTP display a toxic phenotype that can be relieved by depletion of various components of the SAGA complex. Our data provide the first solid evidence for the nuclear target of the IL-1alpha precursor and suggest its novel function in transcriptional control.
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http://dx.doi.org/10.1074/jbc.M306342200DOI Listing
February 2004