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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 217 records · Page 12Linked to original sources

bioRxiv2018

A stable mode of bookmarking by TBP recruits RNA Polymerase II to mitotic chromosomes

How a cell maintains transcriptional fidelity across mitosis has remained an enduring mystery in biology. One challenge arises during mitosis when chromatin becomes condensed and transcription is shut off. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in ES cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes. This stable binding is in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery maintains global transcriptional memory during mitosis.

molecular biology↗

bioRxiv2017

A synthetic biology approach to probing nucleosome symmetry

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.

molecular biology↗

bioRxiv2016

Diatom Centromeres Suggest a Novel Mechanism for Nuclear Gene Acquisition

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. Diatom centromere sequences contain low GC content regions and an abundance of long contiguous AT windows, but lack repeats or other conserved sequence features. Native and foreign sequences of similar GC content can maintain episomes and recruit the diatom centromeric histone protein CENP-A, suggesting non-native sequences can also function as diatom centromeres. Thus, simple sequence requirements enable DNA from foreign sources to incorporate into the nuclear genome repertoire as stable extra-chromosomal episomes, revealing a potential mechanism for bacterial and foreign eukaryotic DNA acquisition.

molecular biology↗

bioRxiv2017

Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in

CRISPR-based gene knock-in at endogenous sites is desirable in multiple fields such as quantitative studies of signal transduction pathways and gene regulation, synthetic biology, and disease modeling. Contrasting the knock-out procedure, a key step of CRISPR knock-in procedure relies on the homology-directed repairing (HDR) process that requires a donor construct as repair template. Therefore, it is desirable to generate a series of donor DNA constructs efficiently and cost-effectively. In this study, we developed a general Gibson assembly procedure that combines strengths of a Modular Overlap-Directed Assembly with Linkers (MODAL) strategy and a restriction enzyme based hierarchical framework. This procedure also allows fusing sgRNAs to the constructs for enhanced homology-directed repairing efficiency. Experimental tests on multiple constructs achieved from 3-8 folds of increase in assembly efficiency to high yield of constructs that failed to make with conventional Gibson assembly. The modularized procedure is simple, fast and cost-effective while making multiple constructs, and a computer package is provided for customized design.

molecular biology↗

bioRxiv2017

Transcriptome-wide analysis of roles for tRNA modifications in translational regulation

Covalent nucleotide modifications in noncoding RNAs such as tRNAs affect a plethora of biological processes, with new functions continuing to be discovered for even well-known tRNA modifications. To systematically compare the functions of a large set of ncRNA modifications in gene regulation, we carried out ribosome profiling and RNA-Seq in budding yeast for 57 nonessential genes involved in tRNA modification. Deletion mutants exhibited a range of translational phenotypes, with modifying enzymes known to modify anticodons, or non-tRNA substrates such as rRNA, exhibiting the most dramatic translational perturbations. Our data build on prior reports documenting translational upregulation of the nutrient-responsive transcription factor Gcn4 in response to numerous tRNA perturbations, and identify many additional translationally-regulated mRNAs throughout the yeast genome. Our data also uncover novel roles for tRNA modifying enzymes in transcriptional regulation of TY retroelements, and in rRNA 2-O-methylation. This dataset should provide a rich resource for discovery of additional links between tRNA modifications and gene regulation.

molecular biology↗

bioRxiv2018

MicroRNA-122 supports robust innate immunity in hepatocytes by suppressing STAT3 phosphorylation

The intrinsic innate immunity of hepatocytes is essential for the control of hepatitis viruses and influences the outcome of antiviral therapy. MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and is a central player in liver biology and disease. However, little is known about the role of miR-122 in hepatocyte innate immunity. Herein, we show that restoring miR-122 levels in hepatoma cells markedly increased the activation of both type III and type I interferons (IFNs) in response to hepatitis C virus (HCV) RNA or poly(I:C). We determined that miR-122 promotes IFN production through down-regulating the tyrosine (Tyr705) phosphorylation of STAT3. We show that STAT3 represses IFN activation by inhibiting interferon regulatory factor 1 (IRF1), which is rate-limiting for maximal IFN expression, especially type III IFNs. Through large-scale screening, we identified that miR-122 targets MERTK, FGFR1 and IGF1R, three oncogenic receptor tyrosine kinases that directly promote STAT3 phosphorylation. These findings reveal a previously unknown role for miR-122 in hepatic immunity and indicate a new potential strategy for treating hepatic infections through targeting STAT3.

molecular biology↗

bioRxiv2017

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Iinenteractions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual or {beta} subunits or as {beta} heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

molecular biology↗

bioRxiv2018

Down-regulated Long Noncoding RNA HOXA11-AS affects trophoblast cell proliferation and migration by regulating RND3 and HOXA7 expression in preeclampsia

The long noncoding RNA HOXA11-AS reveals abnormal expression in numerous human diseases. However, its function and biological mechanisms remain unclear in Preeclampsia (PE). In this study, we report that HOXA11-AS was significantly downregulated in preeclampsic placental tissues and could contribute to the occurrence and development of Preeclampsia. Silencing of HOXA11-AS expression could significantly suppress trophoblast cell growth and migration, whereas HOXA11-AS overexpression facilitated cell growth in HTR-8/SVneo, JEG3 and JAR cell lines. RNA-seq analysis also indicated that HOXA11-AS silencing preferentially regulated numerous genes associated with cell proliferation and cell migration. Mechanistic analyses showed that HOXA11-AS could recruit Ezh2 and Lsd1 protein, and regulate RND3 mRNA expression in nucleus. In cytoplasm, HOXA11-AS modulate HOXA7 expression by sponged miR-15b-5p, thus affecting trophoblast cell proliferation. Together, these resulting data confirm that aberrant expression of HOXA11-AS is involved in the occurrence and development of Preeclampsia, and may act as a prospective diagnosis and therapeutic target in PE.

molecular biology↗

bioRxiv2018

HDAC11 Suppresses the Thermogenic Program of Adipose Tissue via BRD2

Little is known about the biological function of histone deacetylase 11 (HDAC11), which is the lone class IV HDAC. Here, we demonstrate that deletion of HDAC11 in mice stimulates brown adipose tissue (BAT) formation and beiging of white adipose tissue (WAT). Consequently, HDAC11-deficient mice exhibit dramatically enhanced thermogenic potential and, in response to high fat feeding, attenuated obesity, insulin resistance, and hepatic steatosis. Ex vivo and cell-based assays revealed that HDAC11 catalytic activity suppresses the BAT transcriptional program, in both the basal state and in response to {beta}-adrenergic receptor signaling, through a mechanism that is dependent on physical association with BRD2, a bromodomain and extraterminal (BET) acetyl-histone binding protein. These findings define a novel epigenetic pathway for the regulation of energy homeostasis, and suggest potential for HDAC11-selective inhibitors for the treatment of obesity and diabetes.

molecular biology↗

bioRxiv2017

A marker-free co-selection strategy for high efficiency human genome engineering

Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing the levels of gene editing in cell populations remain highly desirable due to the variable efficiency between distinct genomic loci and cell types. Harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems, we designed a simple and robust co-selection strategy for enriching cells harboring either nuclease-driven non-homologous end joining (NHEJ) or homology-directed repair (HDR) events. Selection for dominant alleles of the endogenous sodium-potassium pump (Na+,K+-ATPase) that render cells resistant to ouabain is used to enrich for custom modifications at another unlinked locus of interest, effectively increasing the recovery of engineered cells. The process was readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells (HSPCs). The use of universal CRISPR reagents and a commercially available small molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

molecular biology↗

bioRxiv2018

Enhancing microRNA activity through increased endosomal release mediated by nigericin

The therapeutic promise of small RNA therapeutics (siRNAs, miRNAs) is not only limited by the lack of delivery vehicles, but also by the inability of the small RNAs to reach intracellular compartments where they can be biologically active. We previously reported successful delivery of functionally active miRNAs via receptor-mediated endocytosis1. This type of targeted therapy still faces one of the major challenges in the delivery field, endosomal sequestration. Here, a new method has been developed to promote endosomal escape of delivered miRNA. The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm: early endosomes are rich in sodium ions (Na+) while the intracellular fluid is rich is potassium ions (K+). Exploiting this difference through favoring the influx of K+ into the endosomes without the exchange for a osmotically active ion (Na+), results in an osmotic differential leading to endosome swelling and bursting. One molecule that is able to exchange K+ for an osmotically inactive hydrogen ion is the ionophore nigericin. Via generating an intramolecular miRNA delivery vehicle, containing a ligand, in this case folate, and nigericin we achieve escape of folate-RNA conjugates (e.g. FolamiRs) from their entrapping endosomes into the cytoplasm where they bind the RNA Induced Silencing complex (RISC) and activate the RNAi response.

molecular biology↗

bioRxiv2018

Acute Psychological Stress Triggers Circulating Cell-Free Mitochondrial DNA

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, psychological stress triggers elevation in circulating cell- free mtDNA (ccf-mtDNA). Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibit a 2-3-fold increase in ccf-mtDNA, with no change in nuclear DNA levels, establishing the magnitude and specificity to ccf-mtDNA. In cell-based studies, we show that glucocorticoid signaling - a consequence of psychological stress in humans - is sufficient to induce mtDNA extrusion in a time frame consistent with human psychophysiology. Collectively, these findings provide the first evidence that psychological stress induces ccf-mtDNA and implicate glucocorticoid signaling as a trigger for ccf-mtDNA release. Further work is needed to examine the functional significance of psychological stress-induced ccf-mtDNA as a mitokine in humans.

molecular biology↗

bioRxiv2018

Isw2 and Ino80 chromatin remodeling factors regulate chromatin, replication, and copy number at the yeast ribosomal DNA locus

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA Polymerases I and III, transcription of non-coding RNAs by RNA Polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but relatively little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors affects the occupancy of nucleosomes in the 35S ribosomal RNA gene and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.\n\nAuthor SummaryTo satisfy high cellular demand for ribosomes, genomes contain many copies of the genes encoding the RNA components of ribosomes. In the budding yeast Saccharomyces cerevisiae, these ribosomal RNA genes are located in the \"ribosomal DNA locus\", a highly repetitive array that contains approximately 150 copies of the same unit, in contrast to the single copies that suffice for most genes. This repetitive quality creates unique regulatory needs. Chromatin structure, the packaging and organization of DNA, is a critical determinant of DNA-dependent processes throughout the genome. ATP-dependent chromatin remodeling factors are important regulators of chromatin structure, and yet relatively little is known about how members of this class of protein affect DNA organization or behavior at the rDNA. In this work, we show that the Isw2 and Ino80 chromatin remodeling factors regulate two features of chromatin structure at the rDNA, the occupancy and the positioning of nucleosomes. In addition, we find that these factors regulate two critical processes that function uniquely at this locus: DNA replication originating from within the rDNA array, and the regulated increase of rDNA repeat copy number.

molecular biology↗

bioRxiv2018

AQ-seq: Accurate quantification of microRNAs and their variants

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here we present a high-throughput protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which removes the ligation bias during library preparation. By accurately measuring miRNAs and their variants (known as isomiRs), we identify alternatively processed miRNAs and correct the 5' end usage and strand preference that have been misannotated. We also uncover highly modified miRNAs that are uridylated and adenylated. Taken together, AQ-seq reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

molecular biology↗

bioRxiv2018

Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

Recent studies regarding the origin of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: The Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly-related to life on Earth indicating a shared-ancestry due to lithological exchange, or (2) is unrelated to life on Earth suggesting a convergent origin of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results equivalent to 1 ppb life-detection sensitivity achieved by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier.

molecular biology↗

bioRxiv2018

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

BackgroundHydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs.\n\nResultsHere, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies.\n\nConclusionsThis is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.

molecular biology↗

bioRxiv2018

(-)-Epigallocatechin-3-gallate inhibition of Epstein-Barr virus lytic replication involves latent membrane protein 1-mediated MAPK signaling pathways

AbstractEBV lytic replication has been shown to be important for carcinogenesis. Latent membrane protein 1 (LMP1) plays an important role in the viral latent infection and is abundantly expressed after EBV entry into the lytic cycle. However, the biological significance of LMP1 continuous expression in EBV lytic cycle is still not completely understood. We found that LMP1 promotes EBV reactivation by activating the downstream MAPK signaling in both AGS-EBV and B95.8 cells. In AGS-EBV cells, LMP1 induces EBV the initiation of the EBV lytic cycle in a p53 dependent manner. Activation of c-Jun by LMP1 through JNKs appears to be involved in EBV reactivation in p53 mutant B95.8 cells. We also demonstrated that EGCG, an anti-EBV agent, inhibits LMP1 expression and the activation of the downstream MAPK signaling pathways, followed by downregulation of EBV lytic protein expression level. Together, this study provides the first evidence that LMP1 promotes EBV reactivation via activation of the MAPK signaling pathways. Our findings further demonstrate that the mechanisms underlying EGCG inhibition of the EBV lytic replication involve the suppression of LMP1-mediated MAPK signaling pathways.\n\nSummary statementThis study definitely confirms the role of LMP1 in EBV reactivation and further explores the mechanism by which EGCG inhibits EBV lytic replication.

molecular biology↗

bioRxiv2018

Toward single particle reconstruction without particle picking: Breaking the detection limit

Single-particle cryo-electron microscopy (cryo-EM) has recently joined X-ray crystallography and NMR spectroscopy as a high-resolution structural method for biological macromolecules. In a cryo-EM experiment, the microscope produces images called micrographs. Projections of the molecule of interest are embedded in the micrographs at unknown locations, and under unknown viewing directions. Standard imaging techniques first locate these projections (detection) and then reconstruct the 3-D structure from them. Unfortunately, high noise levels hinder detection. When reliable detection is rendered impossible, the standard techniques fail. This is a problem especially for small molecules, which can be particularly hard to detect. In this paper, we propose a radically different approach: we contend that the structure could, in principle, be reconstructed directly from the micrographs, without intermediate detection. As a result, even small molecules should be within reach for cryo-EM. To support this claim, we setup a simplified mathematical model and demonstrate how our autocorrelation analysis technique allows to go directly from the micrographs to the sought signals. This involves only one pass over the micrographs, which is desirable for large experiments. We show numerical results and discuss challenges that lay ahead to turn this proof-of-concept into a competitive alternative to state-of-the-art algorithms.

molecular biology↗