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bioRxiv2017

Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non-canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.\n\nHighlightsO_LIMeasurement of spliceosome-bound precursor and intermediate in three species\nC_LIO_LINon-canonical splicing events revealed\nC_LIO_LIStatistical modeling uncovers substrate features that predict catalytic efficiency\nC_LIO_LIDiscard of suboptimal substrates occurs in vivo and predicts intron-retained mRNAs\nC_LI

molecular biology↗

bioRxiv2018

4-hydroxyphenylpyruvate dioxygenase thermolability is responsible for temperature-dependent melanogenesis in Aeromonas salmonicida subsP salmonicida

Aeromonas salmonicida subsp. salmonicida (A.s.s) is a major pathogen affecting fisheries worldwide. It is a well-known member of the pigmented Aeromonas species, which produces melanin at [≤] 22 {degrees}C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30-35 {degrees}C while bacterial growth is not affected. The mechanism and biological significance of this temperature-dependent melanogenesis are not clear. Heterologous expression of an A.s.s. 4-hydroxyphenylpyruvate dioxygenase (HppD), the most crucial enzyme in the HGA-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli, suggesting that HppD plays a key role in this process. In the current study, we demonstrated that the extreme thermolability of HppD is responsible for the temperature-dependent melanization of A.s.s. Substitutions in three residues, Ser18, Pro103, or Leu119 of HppD from A.s.s increases the thermolability of this enzyme and results in temperature-independent melanogenesis. Moreover, replacing the corresponding residues of HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A.s.s HppD leads to thermosensitive melanogenesis. Structural analysis suggested that mutations at these sites, especially at position P103, can strengthen the secondary structure of HppD and greatly improve its thermal stability. In addition, we found that HppD sequences of all A.s.s isolates are identical and that two of the three residues are completely conserved within A.s.s isolates, which clearly distinguishes these from other Aeromonas strains. We suggest that this property represents an adaptive strategy to the psychrophilic lifestyle of A.s.s.\n\nImportanceAeromonas salmonicida subsp. salmonicida (A.s.s) is the causative agent of furunculosis, a bacterial septicemia of cold water fish of the Salmonidae family. As it has a well-defined host range, A.s.s has become an ideal model to investigate the co-evolution of host and pathogen. For many pathogens, melanin production is associated with virulence. Although other species of Aeromonas can produce melanin, A.s.s is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here we demonstrate that thermosensitive melanogenesis in A.s.s strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). The strictly conserved hppD sequences among A.s.s and the exclusive thermosensitive pigmentation of these strains might provide insight into the role of melanin in the adaptation to a particular host, and offer a novel molecular marker to readily differentiate A.s.s strains from other A. salmonicida subspecies and Aeromonas species.

molecular biology↗

bioRxiv2026

Elucidating the functional domain architecture of ArCS1, a biomineralizing myosin chitin synthase: I. The role of lipids

In molluscs, chitin synthases are essential for biologically controlled biomineralization, with some variants possessing a myosin motor domain that may link polymer synthesis to the cytoskeleton. Experimentally, we established a reliable workflow for expressing ArCS1_E22TM in Dictyostelium discoideum and developed effective purification methods to reconstitute ArCS1_E22TM in nanodiscs using MSPs and specific lipid composition. MSP1D1deltaH5 proved optimal for nanodisc formation, yielding homogeneous, monodisperse discs (~8.2 nm). Lipids were refined to POPC:POPE:POPG (3:1:1) with 20% cholesterol, improving nanodisc quality and uniformity as observed by negative-stain EM. The full-length ArCS1 and its subdomains were modeled using AlphaFold3; the myosin motor, glycosyltransferase, and transmembrane regions are well-defined internally but loosely constrained relative to one another, suggesting flexible linking and conformational coupling. Modelling with Mg2+ and oleic acid as ligands and comparative analyses with bacterial cellulose synthase and yeast chitin synthase 1 provided insights into substrate binding and a potential mechanism for chitin polymerization and translocation. This research establishes a standard procedure for comprehensive structural analyses of recombinant molluscan chitin synthase in near-native or biomimetic membranes. This sets the stage for high-resolution cryo-electron microscopy to determine the first experimentally resolved structure of a molluscan chitin synthase and to provide insight into the enzyme's architecture and the regulatory mechanisms of biomineralization.

molecular biology↗

bioRxiv2018

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the development of RNA-interactome capture (RNA-IC). However, it remains unknown how responsive is the RBPome and whether these responses are biologically relevant. To answer these questions, we created comparative RNA-IC to analyse cells challenged with an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA binding upon SINV infection are caused by changes in the subcellular localisation of RBPs and RNA availability. Moreover, RBPome responses are crucial, as perturbation of dynamic RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral factor. Therefore, RBPome remodelling provides a mechanism by which cells can extensively rewire gene expression in response to physiological cues.\n\nHIGHLIGHTSO_LIA quarter of the RBPome remodels upon SINV infection.\nC_LIO_LIThe remodelling is caused by changes in protein localisation and RNA availability.\nC_LIO_LIRewiring of the RBPome is crucial for viral infection efficacy.\nC_LIO_LIWe discover RBPs with previously unknown anti- or pro-viral activity.\nC_LI

molecular biology↗

bioRxiv2017

A checkpoint roadmap for the complex cell division of Apicomplexa parasites

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.

cell biology↗

bioRxiv2017

Recent improvements to the automatic characterization and data collection algorithms on MASSIF-1

Macromolecular crystallography (MX) is now a mature and widely used technique essential in the understanding of biology and medicine. Increases in computing power combined with robotics have enabled not only large numbers of samples to be screened and characterised but also for better decisions to be taken on data collection itself. This led to the development of MASSIF-1 at the ESRF, the worlds first beamline to run fully automatically while making intelligent decisions taking user requirements into account. Since opening in late 2014 the beamline has now processed over 39,000 samples. Improvements have been made to the speed of the sample handling robotics and error management within the software routines. The workflows initially put in place, while highly innovative at the time, have been expanded to include increased complexity and additional intelligence using the information gathered during characterisation, this includes adapting the beam diameter dynamically to match the diffraction volume within the crystal. Complex multi-position and multi-crystal data collections are now also integrated into the selection of experiments available. This has led to increased data quality and throughput allowing even the most challenging samples to be treated automatically.\n\nSynopsis\n\nSignificant improvements in the sample location, characterisation and data collection algorithms on the autonomous ESRF beamline MASSIF-1 are described. The workflows now include dynamic beam diameter adjustment and multi-position and multi-crystal data collections.

molecular biology↗

bioRxiv2018

Study on the Difference of Prokaryotic Flora Structure on the Surface of Micro-nano Coating with Different Antifouling Property

Low surface energy composite antifouling coatings prepared from carbon nanotubes (CNTs) and polydimethylsiloxane (PDMS) have good values for investigation of biofouling-related biological questions on marine biofilm. In order to deeply study the mechanism of antifouling on the surface of CNTs-PDMS coatings, it is necessary to investigate the structure of the microbial flora in the early biofilm on the coating surface. In the present study, the specific aim of this study was to investigate the structure differences of prokaryotic flora in biofilm samples at the early stage of biofouling through 16S rDNA based high-throughput DNA sequencing. By annotating high-throughput DNA sequencing results, this study identified dominant prokaryotic phyla and genera in biofilms of CNTs-PDMS coatings and identified significant differences in microbial composition and its dynamics among different coatings. Though the analysis of the Shannon index, Simpson index, Chao1 index and ACE index, coatings with better antifouling properties and antifouling properties have significant differences in community diversity and abundance, indicating different antifouling properties can affect the type and content of biofilm communities. According to the canonical correspondence analysis (CCA), time and temperature are more related to microbial community distribution, while the diameter and length of nanomaterials are less correlated. Through this study, the differences in microbial composition and content of prokaryotic communities, differences in diversity and abundance of sample communities, the differences between multiple samples and the correlation with important environmental factors were preliminarily analyzed, which laid a decent foundation for further research on the mechanism of anti-fouling on the surface of CNTs and PDMS coatings.

molecular biology↗

bioRxiv2017

PrPSc-induced conformational changes and strain-specific structures of PrPSc revealed by Disulfide-crosslink scanning

There exist many phenotypically-varied prion strains, like viruses, despite the absence of conventional genetic material which codes the phenotypic information. As prion is composed solely of the pathological isoform (PrPSc) of prion protein (PrP), the strain-specific traits are hypothesized to be enciphered in the structural details of PrPSc. Identification of the structures of PrPSc is therefore vital for the understanding of prion biology, though they remain unidentified due to the incompatibility of PrPSc with conventional high-resolution structural analyses. Based on our previous hypothesis that the region between the first and the second -helix (H1[~]H2) and the distal region of the third helix (Ctrm) of the cellular isoform of PrP (PrPC) have important roles for efficient interactions with PrPSc, we created series of mutant PrPs with two cysteine substitutions (C;C-PrP) which were systematically designed to form an intramolecular disulfide crosslink between H1[~]H2 and Ctrm and assessed their conformational changes by prions: Specifically, a cysteine substitution in H1[~]H2 from 165 to 169 was combined with cysteine-scanning along Ctrm from 220 to 229. C;C-PrPs with the crosslinks were expressed normally with the similar glycosylation patterns and subcellular localization as the wild-type PrP albeit with varied expression levels. Interestingly, some of the C;C-PrPs converted to the protease-resistant isoforms in the N2a cells persistently infected with 22L prion strain, whereas the same mutants did not convert in the cells infected with another prion strain Fukuoka1, indicating that local structures of PrPSc in these regions vary among prion strains and contribute to prion-strain diversity. Moreover, patterns of the crosslinks of the convertible C;C-PrPs implied drastic changes in positional relations of H1[~]H2 and Ctrm in the PrPSc-induced conformational changes by 22L prion. Thus, disulfide-crosslink scanning is a useful approach for investigation of strain-specific structures of PrPSc, and would be applicable to other types of amyloids as well.

molecular biology↗

bioRxiv2017

Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four dimensional imaging

EDITORIAL SUMMARYThis protocol describes how to estimate and spatially resolve the concentration and copy number of fluorescently tagged proteins in live cells using fluorescence imaging and fluorescence correlation spectroscopy (FCS).\n\nTWEETDetermining protein concentrations and copy numbers in live cells using fluorescence correlation spectroscopy (FCS)-calibrated imaging.\n\nCOVER TEASER Map protein concentrations with FCS-calibrated imagingUp to four primary research articles where the protocol has been used and/or developed:\n\nO_LIWalther, N., Hossain, M. J., Politi, A. Z., Koch, B., Kueblbeck, M., Oedegaard-Fougner, O., Lampe, M. and J. Ellenberg (2018). A quantitative map of human Condensins provides new insights into mitotic chromosome architecture. bioRxiv, 237834. https://doi.org/10.1101/2378342.\nC_LIO_LICai, Y., Hossain, M. J., Heriche, J.-K., Politi, A. Z., Walther, N., Koch, B., Wachsmuth, M., Nijmeijer, B., Kueblbeck, M., Martinic, M., Ladurner, R., Peters, J.M. and J. Ellenberg (2017). An experimental and computational framework to build a dynamic protein atlas of human cell division. bioRxiv, 227751 https://doi.org/10.1101/227751\nC_LIO_LIGermier, T., Kocanova, S., Walther, N., Bancaud, A., Shaban, H.A., Sellou, H., Politi, A.Z., Ellenberg, J., Gallardo, F. and K. Bystricky (2017). Real-Time Imaging of a Single Gene Reveals Transcription-Initiated Local Confinement. Biophysical Journal, 113(7), 1383-1394, https://doi.org/10.1016/j.bpj.2017.08.014.\nC_LIO_LICuylen, S., Blaukopf, C., Politi, A. Z., Muller-Reichert, T., Neumann, B., Poser, I., Ellenberg, J., Hyman, A.A., and D.W. Gerlich (2016). Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature, 535(7611), 308-312. http://doi.org/10.1038/nature18610.\nC_LI\n\nAbstractThe ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables the quantitative understanding of protein dynamics at the physiological level. Genome editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool to investigate spatial and temporal protein dynamics, however it lacks the required quantitative power to make absolute and comparable measurements required for systems analysis. Fluorescence correlation spectroscopy (FCS) on the other hand provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius and oligomerization but lacks the ability for high-throughput application in 4D spatial and temporal imaging. Here, we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high-throughput. These data is processed to yield a calibration curve relating the fluorescence intensities of image voxels to absolute protein abundance. The calibration curve allows the conversion of the arbitrary fluorescence intensities to protein amounts for all voxels of 4D imaging stacks. With our workflow the users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared to other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 day.

molecular biology↗

bioRxiv2018

An EST-SSR based genetic linkage map and identification of QTLs for anthracnose disease resistance in water yam (Dioscorea alata L.)

Water yam (Dioscorea alata L.) is one of the most important food yams with wide geographical distribution in tropics. One of the major constraints to water yam production is anthracnose disease caused by a fungus, Colletotrichum gloesporioides (Penz.). There are no economically feasible solutions as chemical sprays or cultural practices, such as crop rotation are seldom convenient for smallholder farmers for sustainable control of the disease. Breeding for development of durable genetic resistant varieties is known to offer lasting solution to control endemic disease threats to crop production. However, breeding for resistance to anthracnose has been slow considering the biological constraints related to the heterozygous and vegetative propagation of the crop. The development of saturated linkage maps with high marker density, such as SSRs, followed by identification of QTLs can accelerate the speed and precision of resistance breeding in water yam. A total of 380 EST-SSRs were used to generate a saturated linkage map. About 60.19% of SSRs showed Mendelian segregation pattern, however, it had no effect on the construction of linkage map. All 380 EST-SSRs were mapped into 20 linkage groups covering a total length of 2559.66 cM, which agrees with the diploid nature (2n = 2x = 20) of the parents used in the cross. Majority of the markers were mapped on linkage group 1 comprising of 97 EST-SSRs. This is the first genetic linkage map of water yam constructed using EST-SSRs. QTL localization was based on phenotypic data collected over a 3-year period of inoculating the mapping population with the most virulent strain of C. gloeosporoides from West Africa. Using the mean permutation value of LOD scores as threshold value for declaring a putative QTL on all linkage groups, one QTL was consistently observed on linkage group (LG) 14 in all the three years and average score data. This QTL was found at position interval of 71.12 - 84.76 cM explaining 68.94% of the total phenotypic variation in the average score data. The high marker density allowed identification of QTLs and association for anthracnose disease, which could be validated in other mapping populations and used in marker-assisted breeding in D. alata improvement programmes.

molecular biology↗

bioRxiv2017

AtRsgA from Arabidopsis thaliana controls maturation of the small subunit of the chloroplast ribosome

SummaryPlastid ribosomes are very similar in structure and function to ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favourable at biological conditions, it requires activity of many assembly factors. Here, we have characterized a homolog of bacterial rsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA-sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were upregulated, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signalling pathways. To conclude, this study reveals a new chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.\n\nSignificance statementAtRsgA is an assembly factor necessary for maturation of the small subunit of the chloroplast ribosome. Depletion of AtRsgA leads to dwarfed, chlorotic plants and smaller, but more numerous chloroplasts. Large-scale transcriptomic and proteomic analysis revealed that chloroplast-encoded and - targeted proteins were less abundant, while the corresponding transcripts were upregulated in the mutant. We analyse the transcriptional responses of several retrograde signalling pathways to suggest a mechanism underlying this compensatory response.

molecular biology↗

bioRxiv2016

Validation of Reference Genes for RT-qPCR in Marine Bivalve Ecotoxicology: Systematic Review and Case Study

Reverse transcription real-time quantitative PCR (RT-qPCR) is the predominant method of choice for the quantification of mRNA transcripts of a selected gene of interest. Here reference genes are commonly used to normalize non-biological variation in mRNA levels and their appropriate selection is therefore essential for the accurate interpretation the collected data. In recent years the use of multiple validated references genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to experimentally validate putative reference genes under specific experimental conditions, determine the optimal number of reference genes to be employed, and report the method or methods used.\n\nUnder this premise, we assessed the current state of reference gene base normalization in RT-qPCR bivalve ecotoxicology studies (post 2011), employing a systematic quantitative literature review. A total of 52 papers published met our criteria and were analysed for the gene or genes used, whether they employed multiple reference genes, as well as the validation method employed. In addition we performed a case study using primary hemocytes from the marine bivalve Ruditapes philippinarum after in vitro copper exposure. Herein we further critically discuss methods for reference gene validation, including the established algorithms geNorm, NormFinder and BestKeeper, as well as the popular online tool RefFinder.\n\nWe identified that RT-qPCR normalization in bivalve ecotoxicology studies is largely performed using single reference genes, while less than 40% of the studies attempted to experimentally validate the expression stability of the reference genes used. 18s rRNA and {beta}-Actin were the most popular genes, yet their un-validated use did introduce artefactual variance that altered the interpretation of the resulting data, while the use of appropriately validated reference genes did substantially improve normalization. Our findings further suggest that combining the results from multiple individual algorithms and calculating the overall best-ranked gene, as e.g. computed by the RefFinder tool, does not by default lead to the identification of the most suitable reference gene or combination of reference genes.

Molecular Biology↗

bioRxiv2018

Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

Infections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

molecular biology↗

bioRxiv2018

Hydroxymethylated-P16 Allele Is Transcription-Inactive

Background5-Methylcytosine can be oxidized into 5-hydroxymethylcytosine (5hmC) in the genome. Methylated-P16 (P16M) can be oxidized into completely hydroxymethylated-P16 (P16H) in human cancer and precancer cells. The aim of this study is to investigate the biological function of P16H.\n\nMethodsTrue P16M and P16H were analyzed using bisulfite/TAB-based assays. A ZFP-based P16-specific dioxygenase (P16-TET) was constructed and used to induce P16H. Cell proliferation and migration were determined with a series of biological analyses.\n\nResults(A) The 5hmCs were enriched in the antisense-strand of the P16 exon-1 in HCT116 and AGS cells containing methylated-P16 alleles (P16M). (B) P16-TET induced both P16H and P16 demethylation in H1299 and AGS cells and reactivated P16 expression. Notably, P16H was only detectable in the sorted P16-TET H1299 and AGS cells that did not show P16 expression. (C) P16-TET significantly inhibited the xenograft growth derived from H1299 cells in NOD-SCID mice, but did not inhibit the growth of P16-deleted A549 control cells. P16-siRNA knockdown could rescue P16-TET-inhibited cell migration.\n\nConclusionHydroxymethylated P16 alleles are transcriptionally inactive.\n\nAUTHOR SUMMARYIt is well known that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidations, a certain proportion of 5hmCs remain in the genome. Although it is supposed that occurrence of 5hmCs may contribute to the flexibility of chromatin and the protection of the bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In the present study, we engineered a zinc-finger protein-based P16-specific DNA dioxygenase and used it to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that the hydroxymethylated P16 alleles retain transcriptionally inactive. This is supported by our recent findings that mRNAs are always transcribed only from the unmethylated P16 strands, but not from the hydroxymethylated/methylated strands in HCT116 cells, and that the risks for malignant transformation are similar for patients with the P16 methylation-positive oral epithelial dysplasia with and without P16 hydroxymethylation in a prospective study.

molecular biology↗

bioRxiv2017

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.

molecular biology↗

bioRxiv2018

Changes in pregnancy-related serum biomarkers early in gestation are associated with later development of preeclampsia

BackgroundPlacental protein expression plays a crucial biological role during normal and complicated pregnancies. We hypothesized that: (1) circulating pregnancy-associated, placenta-related protein levels throughout gestation reflect the uncomplicated, full-term temporal progression of human gestation, and effectively estimates gestational ages (GAs); (2) pregnancies with underlying placental pathology, such as preeclampsia (PE), are associated with disruptions in this GA estimation in early gestation; (3) malfunctions of this GA estimation can be employed to identify impending PE. In addition, to explore the underlying biology and PE etiology, we set to compare protein gestational patterns of human and mouse, using pregnant heme oxygenase-1 (HO-1) heterozygote (Het) mice, a mouse model reflecting PE-like symptoms.\n\nMethodsSerum levels of circulating placenta-related proteins - leptin (LEP), chorionic somatomammotropin hormone like 1 (CSHL1), elabela (ELA), activin A, soluble fms-like tyrosine kinase 1 (sFlt-1), and placental growth factor (PlGF)- were quantified by ELISA in blood serially collected throughout human pregnancies (20 normal subjects with 66 samples, and 20 PE subjects with 61 samples). Linear multivariate analysis of the targeted serological protein levels was performed to estimate the normal GA. Logarithmic transformed mean-squared errors of GA estimations were used to identify impending PE. Then the human gestational protein patterns were compared to those in the pregnant HO-1 mice.\n\nResultsAn elastic net (EN)-based gestational dating model was developed (R2 = 0.76) and validated (R2 = 0.61) using the serum levels of the 6 proteins at various GAs from women with normal uncomplicated pregnancies (n = 10 for training and n = 6 for validation). In pregnancies complicated by PE (n = 14), the EN model was not (R2 = -0.17) associated with GA at sampling in PE. Statistically significant deviations from the normal GA EN model estimations were observed in PE-associated pregnancies between GAs of 16-30 weeks (P = 0.01). The EN model developed with 5 proteins (ELA excluded due to the lack of robustness of the mouse ELA essay) performed similarly on normal human (R2 = 0.68) and WT mouse (R2 = 0.85) pregnancies. Disruptions of this model were observed in both human PE-associated (human: R2 = 0.27) and mouse HO-1 Het (mouse: R2 = 0.30) pregnancies. LEP out performed sFlt-1 and PlGF in differentiating impending PE at early human and late mouse gestations.\n\nConclusionsAs revealed in both human and mouse GA EN analyses, temporal serological placenta-related protein patterns are tightly regulated throughout normal human pregnancies and can be significantly disrupted in pathologic PE states. LEP changes earlier during gestation than the well-established late GA PE biomarkers (sFlt-1 and PlGF). Our HO-1 Het mouse analysis provides direct evidence of the causative action of HO-1 deficiency in LEP upregulation in a PE-like murine model. Therefore, longitudinal analyses of pregnancy-related protein patterns in sera, may not only help in the exploration of underlying PE pathophysiology but also provide better clinical utility in PE assessment.

molecular biology↗

bioRxiv2013

Differentiation-dependent telomeric long non-coding transcription in a model of skeletal myogenesis

Telomeres comprise the distal ends of eukaryotic chromosomes, serve to maintain genomic integrity and are extended by the ribonucleoprotein telomerase. Recent evidence indicates that telomeres are transcribed to generate long non-coding RNAs (lncRNAs) and that these transcripts (TERRA) may inhibit telomerase activity. In this study we assessed telomerase activity and telomeric lncRNA expression in a mouse model of skeletal myogenesis. Using the C2C12 cell line we demonstrated decreased telomerase activity during differentiation into terminally-differentiated skeletal myotubes. Despite existing in a post-mitotic state, residual telomerase activity remained in C2C12 myotubes, indicating a role independent of telomere extension. Telomeric transcripts were detected in both myoblasts and myotubes, with reduced expression during differentiation correlating with reduced telomerase expression. Our data indicate that in a mouse model of skeletal myogenesis TERRA expression does not reduce telomerase activity, suggesting that their relationship is more complex than originally perceived; the role of telomeric derived lncRNAs in relation to telomerase activity may be cell-type specific. These findings raise the possibility for novel non-telomerase regulatory function for TERRA-lncRNAs during skeletal myogenesis.

Molecular Biology↗

bioRxiv2014

How and where to look for tRNAs in Metazoan mitochondrial genomes, and what you might find when you get there

The ability to locate and annotate mitochondrial genes is an important practical issue, given the rapidly increasing number of mitogenomes appearing in the public databases. Unfortunately, tRNA genes in Metazoan mitochondria have proved to be problematic because they often vary in number (genes missing or duplicated) and also in the secondary structure of the transcribed tRNAs (T or D arms missing). I have performed a series of comparative analyses of the tRNA genes of a broad range of Metazoan mitogenomes in order to address this issue. I conclude that no single computer program is necessarily capable of finding all of the tRNA genes in any given mitogenome, and that use of both the ARWEN and DOGMA programs is sometimes necessary because they produce complementary false negatives. There are apparently a very large number of erroneous annotations in the databased mitogenome sequences, including missed genes, wrongly annotated locations, false complements, and inconsistent criteria for assigning the 5' and 3' boundaries; and I have listed many of these. The extent of overlap between genes is often greatly exaggerated due to inconsistent annotations, although notable overlaps involving tRNAs are apparently real. Finally, three novel hypotheses were examined and found to have support from the comparative analyses: (1) some organisms have mitogenomic locations that simultaneously code for multiple tRNAs; (2) some organisms have mitogenomic locations that simultaneously code for tRNAs and proteins (but not rRNAs); and (3) one group of nematodes has several genes that code for tRNAs lacking both the D and T arms.

Molecular Biology↗