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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 883 records · Page 49Linked to original sources

bioRxiv2018

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

AbstractDNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

molecular biology↗

bioRxiv2018

Detection and Degradation of Nonsense-mediated mRNA Decay Substrates Involve Two Distinct Upf1-bound Complexes

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

molecular biology↗

bioRxiv2018

Non-histone human chromatin protein, PC4 is critical for genomic integrity and negatively regulates autophagy

Multifunctional human transcriptional positive co-activator 4 (PC4) is a bonafide non-histone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional genome organization. Knockdown of PC4 expression leads to a drastic open conformation of the chromatin and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation possibly through enhanced autophagic activity. PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the survivability PC4 knockdown cells. On the contrary, complementation with wild type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during a stress condition such as radiation.

molecular biology↗

bioRxiv2018

Atomic model of microtubule-bound tau

Tau is a developmentally regulated protein found in axons, whose physiological role is to stabilize and bundle microtubules (MTs). Hyper-phosphorylation of tau is thought to cause its detachment from MTs and subsequent aggregation into pathological fibrils that have been implicated in Alzheimers disease pathogenesis. Despite its known MT binding role, there is no consensus regarding which tau residues are crucial for tau-MT interactions, where on the MT tau binds, and how binding results in MT stabilization. We have used cryo-EM to visualize the interaction of different tau constructs with MTs at high resolution (3.2-4.8 [A]) and used computational approaches to generate atomic models of tau-tubulin interactions. Our work shows that the highly conserved tubulin-binding repeats within tau adopt very similar structures in their interactions with the MT. Each tau repeat binds the MT exterior and adopts an extended structure along the crest of the protofilament (PF), interacting with both - and {beta}-tubulin, thus stabilizing the interface between tubulin dimers. Our structures agree with and explain previous biochemical data concerning the effect of phosphorylation on MT affinity and lead to a model in which tau repeats bind in tandem along a PF, tethering together tubulin dimers and stabilizing longitudinal polymerization interfaces. These structural findings could establish a basis of future treatments aiming at the selective stabilization of tau-MT interactions.

molecular biology↗

bioRxiv2018

Intron-mediated enhancement is not limited to introns

Certain introns strongly increase mRNA accumulation by a poorly understood mechanism known as Intron-Mediated Enhancement (IME). Introns that boost expression by IME have no effect when located upstream of or more than ~1 Kb downstream from the start of transcription. The sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, can convert a non-stimulating intron into one that strongly increases mRNA accumulation. We tested the ability of an intron containing this motif to stimulate expression from different locations and found that it had the same positional requirements as naturally occurring IME introns. The motif also stimulated gene expression from within the 5-UTR and coding sequences of an intronless construct. Furthermore, the 5-UTR of another gene increased expression when inserted into an otherwise non-stimulating intron in coding sequences. These results demonstrate that splicing is not required for intron-mediated enhancement, and suggest that other sequences downstream of the transcription start site in addition to introns may stimulate expression by a similar mechanism.

molecular biology↗

bioRxiv2018

Simultaneous detection of DNA and RNA virus species involved in bovine respiratory disease by PCR-free rapid tagmentation-based library preparation and MinION nanopore sequencing

AbstactThe Oxford Nanopore MinION Mk1B is a portable 90 g device that sequences DNA directly at 450 bases/second generating sequence reads in excess of 400 kb. Recent improvements in error rate and speed of library preparation mean that this device has considerable potential for rapid molecular bovine pathogen diagnostics. We tested the MinION for rapid untargeted detection of viral pathogens associated with bovine respiratory disease (BRD), an economically important disease often involving primary infection of the lung by one or more of a number of DNA and/or RNA viruses. We combined three foetal lung cell cultures which were infected with either Bovine Respiratory Syncytial Virus (BRSV), Bovine Herpes Virus 1 (BoHV1) or Bovine Parainfluenza Virus 3 (BPI-3). BoHV1 is a DNA virus and BPI-3 and BRSV are RNA viruses. The cell cultures were treated with DNase and RNase to deplete bovine nucleic acid prior to viral nucleic acid extraction and double-stranded cDNA synthesis. Sequencing libraries were generated by PCR-free tagmentation in under 10 minutes and loaded onto a MinION sequencer. Approximately 7,000 sequencing reads were generated and analysed using high-throughput local BLAST against the NCBI nr/nt database. The top BLAST hit for 2,937 of these reads was identified as a virus. Of these, 2,926 (99.6%) were correctly identified either as BoHV1, BRSV or BPI-3.

molecular biology↗

bioRxiv2018

CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts

Co-transcriptional capping of RNA polymerase II (Pol II) transcripts by capping enzyme proceeds orders of magnitude more efficiently than capping of free RNA. Previous studies brought to light a role for the phosphorylated Pol II CTD in activation of co-transcriptional capping; however, CTD phosphorylation alone could not account for the observed magnitude of activation. Here, we exploit a defined Pol II transcription system that supports both CTD phosphorylation and robust activation of capping to dissect the mechanism of co-transcriptional capping. Taken together, our findings identify a novel CTD-independent, but Pol II-mediated, mechanism that functions in parallel with CTD-dependent processes to ensure optimal capping, and they support a \"tethering\" model for the mechanism of activation.

molecular biology↗

bioRxiv2018

GTSF-1 is required for the formation of a functional RNA-dependent RNA Polymerase complex in C. elegans

In every domain of life, Argonaute proteins and their associated small RNAs regulate gene expression. Despite great conservation of Argonaute proteins throughout evolution, many proteins acting in small RNA pathways are not widely conserved. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured, acidic C-terminal tail, are conserved in animals and act in small RNA pathways. In fly and mouse, they are required for fertility and have been shown to interact with Piwi clade Argonautes. We identified T06A10.3 as the Caenorhabditis elegans Gtsf1 homolog and named it gtsf-1. Given its conserved nature and roles in Piwi-mediated gene silencing, we sought out to characterize GTSF-1 in the context of the small RNA pathways of C. elegans. Like its homologs, GTSF-1 is required for normal fertility. Surprisingly, we report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show strong depletion of a class of endogenous small RNAs, known as 26G-RNAs, and fully phenocopy mutants lacking RRF-3, the RNA-dependent RNA Polymerase that synthesizes 26G-RNAs. We show, both in vivo and in vitro, that GTSF-1 specifically and robustly interacts with RRF-3 via its tandem CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex, also known as ERIC, thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may similarly act to drive the assembly of larger complexes that subsequently act in small RNA production and/or in imposing small RNA-mediated silencing activities.

molecular biology↗

bioRxiv2018

The exon junction complex regulates the release and phosphorylation of paused RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) plays a critical and conserved role in this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.

molecular biology↗

bioRxiv2018

Transcriptomic analysis of the early strobilar development of Echinococcus granulosus

BackgroundEchinococcus granulosus has a complex life cycle involving two mammalian hosts. The transition from one host to another is accompanied by changes in gene expression, and the transcriptional events that underlie these processes have not yet been fully characterized.\n\nResultsIn this study, RNA-seq is used to compare the transcription profiles of four time samples of E. granulosus protoscoleces in vitro induced to strobilar development. We identified 818 differentially expressed genes, which were divided into eight expression clusters formed over the entire 24 hours time course and indicated different transcriptional patterns. An enrichment of gene transcripts with molecular functions of signal transduction, enzymes and protein modifications was observed with progression of development.\n\nConclusionThis transcriptomic study provides insight for understanding the complex life cycle of E. granulosus and contributes for searching for the key genes correlating with the strobilar development, providing interesting hints for further studies.

molecular biology↗

bioRxiv2018

Delegating Sex: differential gene expression in stolonizing syllids uncover the hormonal control of reproduction in Annelida

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed at the posterior end of the animal. When the stolons mature, they detach from the adult and the gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand the reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and non-reproductive individuals of Syllis magdalena, and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase, vitellogenin, testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase 2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine and serotonin, might trigger the stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis is complete. The process seems to be under circadian control, as indicated by the expression patterns of r-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation, and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization.

molecular biology↗

bioRxiv2018

Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose

Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2'-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2'-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2'-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation.\n\nHIGHLIGHTSO_LIMeTH-seq identifies 2'-O-methylribose genome-wide at single-nucleotide resolution\nC_LIO_LIFive conserved methyltransferases interact with yeast mRNA\nC_LIO_LISpb1 is a major mRNA 2'-O-methyltransferase, and targets most ribosomal protein mRNAs\nC_LIO_LISPB1 expression is required to maintain normal levels of Spb1 target mRNAs\nC_LI

molecular biology↗

bioRxiv2018

An eicosanoid protects from statin-induced myopathic changes in primary human cells

Statin-related muscle side effects are a constant healthcare problem since patient compliance is dependent on side effects. Statins reduce plasma cholesterol levels and can prevent secondary cardiovascular disease. Although statin-induced muscle damage has been studied, preventive or curative therapies are yet to be reported.\n\nWe exposed primary human muscle cell populations (n=25) to a lipophilic (simvastatin) and a hydrophilic (rosuvastatin) statin and analyzed their expressome. Data and pathway analyses included GOrilla, Reactome and DAVID. We measured mevalonate intracellularly and analyzed eicosanoid profiles secreted by human muscle cells. Functional assays included proliferation and differentiation quantification.\n\nMore than 1800 transcripts and 900 proteins were differentially expressed after exposure to statins. Simvastatin had a stronger effect on the expressome than rosuvastatin, but both statins influenced cholesterol biosynthesis, fatty acid metabolism, eicosanoid synthesis, proliferation, and differentiation of human muscle cells. Cultured human muscle cells secreted {omega}-3 and {omega}-6 derived eicosanoids and prostaglandins. The {omega}-6 derived metabolites were found at higher levels secreted from simvastatin-treated primary human muscle cells. Eicosanoids rescued muscle cell differentiation.\n\nOur data suggest a new aspect on the role of skeletal muscle in cholesterol metabolism. For clinical practice, the addition of omega-n fatty acids could be suitable to prevent or treat statin-myopathy.

molecular biology↗

bioRxiv2018

A novel environmental DNA (eDNA) sampling method for aye-ayes from their feeding traces

Non-invasive sampling is an important development in population genetic monitoring of wild animals. Particularly, the collection of environmental DNA (eDNA) which can be collected without needing to encounter the target animal, facilitates the genetic analysis of cryptic and threatened species. One method that has been applied to these types of sample is target capture and enrichment which overcomes the issue of high proportions of exogenous (non-host) DNA from these lower quality samples. We tested whether target capture of mitochondrial DNA from sampled feeding traces of wild aye-ayes would yield mitochondrial DNA sequences for population genetic monitoring. We sampled gnawed wood from feeding traces where aye-ayes excavate wood-boring insect larvae from trees. We designed RNA probes complementary to the aye-ayes mitochondrial genome and used these to isolate aye-aye DNA from other non-target DNA in these samples. We successfully retrieved six near-complete mitochondrial genomes from two sites within the aye-ayes geographic range that had not been sampled previously. This method can likely be applied to alternative foraged remains to sample species other than aye-ayes. Our method demonstrates the application to next-generation molecular techniques to species of conservation concern.

molecular biology↗

bioRxiv2018

α-proteobacterial RNA degradosomes assemble liquid-liquid phase separated RNP bodies

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less \"droplet\" organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS droplets that we term bacterial RNP-bodies (BR-bodies) similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related -proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS droplets, providing an effective strategy for subcellular organization in cells lacking membrane bound compartments.

molecular biology↗

bioRxiv2018

Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations

Protein aggregates such as those formed in neurodegenerative diseases can be degraded via autophagy. To assess changes in autophagic flux in zebrafish models of familial Alzheimers disease (fAD) mutations, we first developed a transgene, polyQ80-GFP-v2A-GFP, expressing equimolar amounts of aggregating polyQ80-GFP and a free GFP internal control in zebrafish embryos and larvae. This assay detects changes in autophagic flux by comparing the relative strength of polyQ80-GFP and free GFP moiety signals on western immunoblots probed with an antibody detecting GFP. However, the assays application is limited by the toxicity of polyQ80-GFP, and because aggregation of this protein may, itself, induce autophagy. To overcome these issues, we subsequently developed a similar ratiometric assay where expression of a GFP-Lc3a-GFP transgene generates initially equimolar amounts of GFP-Lc3a (directed to autophagic degradation) and a free GFP internal control. The sensitivity of this latter assay is reduced by a cellular protease activity that separates Lc3a from GFP-Lc3a, thus contributing to the apparent free GFP signal and somewhat masking decreases in autophagic flux. Nevertheless, the assay demonstrates significantly decreased autophagic flux in zebrafish lacking presenilin2 gene activity supporting that the Presenilin2 protein, like human PRESENILIN1, plays a role(s) in autophagy. Zebrafish heterozygous for a typical fAD-like, reading-frame-preserving mutation in psen1 show decreased autophagic flux consistent with observations in mammalian systems. Unexpectedly, a zebrafish model of the only confirmed reading-frame-truncating fAD mutation in a human PRESENILIN gene, the K115Efs mutation of human PSEN2, shows possibly increased autophagic flux in young zebrafish (larvae).

molecular biology↗

bioRxiv2018

The tumor suppressor FBW7 and the vitamin D receptor are mutual cofactors.

The E3 ligase FBW7 targets drivers of cell cycle progression such as c-MYC for proteasomal degradation. It is frequently mutated in cancer, and is a tumor suppressor. Extensive epidemiological data links vitamin D deficiency to increased incidence of several cancers, although the underlying cancer-preventive mechanisms are poorly understood. Here, we show that hormonal 1,25-dihydroxyvitamin D3 (1,25D) rapidly stimulates the interaction of the VDR with FBW7, and that of FBW7 with c-MYC. In contrast, it blocks the association of FBW7 with c-MYC antagonist MXD1. 1,25D also enhances the association of FBW7, proteasome subunits, and ubiquitin with DNA-bound c-MYC, consistent with induced degradation of c-MYC on DNA. In addition to c-MYC, 1,25D accelerates the turnover of other FBW7 target proteins. Intriguingly, FBW7 is essential for optimal VDR gene expression. It is also recruited to VDR targets genes, and its depletion attenuates 1,25D-stimulated VDR DNA binding, transactivation, and cell cycle arrest. Thus, the VDR and FBW7 are mutual cofactors, which provides a molecular basis for the cancer-preventive actions of vitamin D through accelerated turnover of FBW7 target proteins.

molecular biology↗

bioRxiv2018

Optimized real-time qPCR assays for detecting and quantifying the Fusarium and Microdochium species responsible for wheat head blight, as defined by MIQE guidelines

In order to better understand the Fusarium head blight disease, reliable real-time PCR assays for detection and quantification of fungal species belonging to the Fusarium and Microdochium genus are needed. Specific qPCR assays were developed for nine of those species. All criteria required for reproducing the assays are presented. The assays were species specific and allow quantification of at least 5 pg of fungal DNA and detection of 0.5 pg of fungal DNA per PCR reaction. Moreover we showed that the quantification performances of the tests were not altered in the presence of DNA of closely related species in the sample. The assays were tested on field samples and have been already used in greenhouse experiments.

molecular biology↗