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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Laboratory and Molecular Surveillance of Paediatric Typhoidal Salmonella in Nepal: Antimicrobial Resistance and Implications for Vaccine Policy

BackgroundChildren are substantially affected by enteric fever in most settings with a high burden of the disease, which could be due to immune naivety, or enhanced risk of exposure to the pathogen. Although Nepal is a high burden setting for enteric fever, the bacterial population structure and transmission dynamics are poorly delineated in young children, the proposed target group for immunization programs.\n\nMethodsBlood culture surveillance amongst children aged 2 months to 15 years of age was conducted at Patan Hospital between 2008 and 2016. A total of 198 S. Typhi and 66 S. Paratyphi A isolated from children treated in both inpatient and outpatient settings were subjected to whole genome sequencing and antimicrobial susceptibility testing. Demographic and clinical data were also collected from the inpatients. The resulting data were used to place these paediatric Nepali isolates into a worldwide context, based on their phylogeny and carriage of molecular determinants of antimicrobial resistance (AMR).\n\nResultsChildren aged [≤]4 years made up >40% of the inpatient population. The majority of isolates (78 %) were S. Typhi, comprising several distinct genotypes but dominated by 4.3.1 (H58). Several distinct S. Typhi genotypes were identified, but the globally disseminated S. Typhi clade 4.3.1 (H58) dominated. The majority of isolates (86%) were insusceptible to fluoroquinolones. This was mainly associated with S. Typhi H58 Lineage II and S. Paratyphi A; non-susceptible strains from these two genotypes accounted for 50% and 25% of all enteric fever cases. Multi-drug resistance (MDR) was rare (3.5% of S. Typhi, 0 S. Paratyphi A) and restricted to chromosomal insertions of AMR genes in H58 lineage I strains. Comparison to global data sets showed the local S. Typhi and S. Paratyphi A strains had close genetic relatives in other South Asian countries, indicating regional strain circulation.\n\nConclusionsThese data indicate that enteric fever in Nepal continues to be a major public health issue with ongoing inter- and intra-country transmission, and highlights the need for regional coordination of intervention strategies. The absence of a S. Paratyphi A vaccine is cause for concern, given its prevalence as an enteric fever agent in this setting, and the large proportion of isolates displaying fluoroquinolone resistance. This study also highlights an urgent need for routine laboratory and molecular surveillance to monitor the epidemiology of enteric fever and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions in prevention and control of this febrile illness.

molecular biology↗

bioRxiv2018

How Do Telomeres Block Checkpoint Activation?

Genome instability is a potentially lethal event for a eukaryotic cell, and a mutational force for genetic diseases such as cancer. DNA double-strand breaks (DSBs) can drive genome instability and are sensed by the DNA damage checkpoint, a defined set of evolutionarily-conserved proteins that bind the DSB to signal a pause or arrest of the cell cycle1 and recruit proteins to repair the DNA lesion2,3. Telomeres, the physical ends of linear eukaryotic chromosomes, are specialized DSBs that suppress DNA damage checkpoint activation by an unknown mechanism(s), even though telomeres are bound by many of the DNA damage checkpoint proteins that signal cell cycle arrest4. Carneiro et al. (Nature 467: 228-232) addressed this question using Schizosaccharomyces pombe cells that lack Taz1, the protein that binds to double-stranded telomere repeats5. Te ...

molecular biology↗

bioRxiv2018

The repressive and alleviating nature of FACT shapes the transcriptional landscape in ES cells

The conserved and essential histone chaperone FACT (Facilitates Chromatin Transcription) reorganizes nucleosomes during DNA transcription, replication and repair and ensures both, efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA Polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to up-regulation of pro-proliferative genes and key pluripotency factors concomitant with hyper-proliferation of mES cells. Using MNase-, ATAC-, and Nascent Elongating Transcript Sequencing (NET-seq) we show that up-regulation of genes coincides with loss of nucleosomes upstream of the TSS and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes ES cells for the neuronal lineage.

molecular biology↗

bioRxiv2018

Loss of PRMT5 promotes PDGFRα degradation during oligodendrocyte differentiation and myelination

Platelet derived growth factor receptor (PDGFR) signaling is required for proliferation, commitment and maintenance of oligodendrocyte (OL) precursor cells (OPCs). PDGFR signaling promotes OPC homeostasis and its attenuation signals OPC differentiation and maturation triggering the onset of myelination of the central nervous system (CNS). The initial steps of how PDGFR signaling is attenuated are still poorly understood. Herein we show that decreased Protein Arginine MethylTransferase5 (PRMT5) expression, as occurs during OPC differentiation, is involved in the down-regulation of PDGFR by modulating its cell surface bioavailability leading to its degradation in a Cbldependent manner. Mechanistically, loss of arginine methylation at R554 of the PDGFR intracellular domain reveals a masked Cbl binding site at Y555. Physiologically, depletion of PRMT5 in OPCs results in severe CNS myelination defects. We propose that decreased PRMT5 activity initiates PDGFR degradation to promote OL differentiation. More broadly, inhibition of PRMT5 may be used therapeutically to manipulate PDGFR bioavailability.

molecular biology↗

bioRxiv2018

A conserved mechanism for regulation of endo-lysosomal pH by histone deacetylases

The pH of the endo-lysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover and signaling functions in the cell. Dysregulation of endo-lysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders including Alzheimers disease. Therefore, understanding mechanisms that regulate luminal pH may be key to identifying new targets for treatment of these disorders. Meta-analysis of yeast microarray databases revealed that nutrient limiting conditions upregulated transcription of the endosomal Na+/H+ exchanger Nhx1 by inhibition of the histone deacetylase (HDAC) Rpd3, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of Nhx1 orthologs DmNHE3 and NHE6 respectively, was also mediated by histone deacetylases. We show that NHE6 is a target of cAMP-response element-binding (CREB) protein, providing a molecular mechanism for nutrient and HDAC dependent regulation of endosomal pH. Control of NHE6 expression by pharmacological targeting of the CREB pathway can be used to regulate endosomal pH and restore defective amyloid A{beta} clearance in an ApoE4 astrocyte model of Alzheimers disease. These observations from yeast, fly, mouse and cell culture models reveal an evolutionarily conserved mechanism for regulation of endosomal NHE expression by histone deacetylases and offer new therapeutic strategies for modulation of endo-lysosomal pH in fungal infection and human disease.

molecular biology↗

bioRxiv2018

Valproic acid attenuates hyperglycemia-induced complement and coagulation cascade gene expression

Atherothrombosis remains the leading cause of morbidity and mortality in patients diagnosed with diabetes mellitus, but the molecular mechanisms underpinning this remain unresolved. As the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is an interest in understanding the mechanisms of hepatic cell activation under hyperglycemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycemia stimulates major changes in chromatin organisation and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA; IUPAC: 2-propylpentanoic acid) is able to reverse some of these metabolic changes. In this study, we used deep transcriptome sequencing to show that VPA attenuates hyperglycemia-induced activation of complement and coagulation cascade genes. These findings reveal a novel mechanism of VPA protection against hyperglycemia, which might improve the therapeutic approaches for diabetes.

molecular biology↗

bioRxiv2018

A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

For long-read sequencing applications, shearing of DNA is a significant issue as it limits the read-lengths generated by sequencing. During extraction and storage of DNA the DNA polymers are susceptible to physical and chemical shearing. In particular, the mechanisms of physical shearing are poorly understood in most laboratories as they are of little relevance to commonly used short-read sequencing technologies. This study draws upon lessons learned in a diverse set of research fields to create a comprehensive theoretical framework for obtaining high molecular weight DNA (HMW-DNA) to support improved quality management in laboratories and biobanks for long-read sequencing applications.\n\nUnder common laboratory conditions physical and chemical shearing yields DNA fragments of 5-35 kilobases (kb) in length. This fragment length is sufficient for DNA sequencing using short-read technologies but for Nanopore sequencing, linked reads and single molecular real time sequencing (SMRT) poorly preserved DNA will limit the length of the reads generated.\n\nThe shearing process can be divided into physical and chemical shearing which generates different patterns of fragmentation. Exposure to physical shearing creates a characteristic fragment length where the main cause of shearing is shear stress induced by turbulence. The characteristic fragment length is several thousand base pairs longer than the reads produced by short-read sequencing as the shear stress imposed on short DNA fragments is insufficient to shear the DNA. This characteristic length can be measured using gel electrophoresis or instruments for DNA fragment analysis. Chemical shearing generates randomly distributed fragment lengths visible as a smear of DNA below the peak fragment length. By measuring the peak of the DNA fragment length distribution and the proportion of very short DNA fragments, both sources of shearing can be measured using commonly used laboratory techniques, providing a suitable quantification of DNA integrity of DNA for sequencing with long-read technologies.

molecular biology↗

bioRxiv2018

Comprehensive characterization of transcript diversity at the human NODAL locus

NODAL, a morphogen belonging to the transforming growth factor beta (TG{beta}) superfamily, is essential during embryogenesis where it induces axis formation and left-right asymmetry. NODAL is also required for the maintenance of human embryonic stem cell pluripotency, and emerges in many cancer types concomitant with metastasis and therapy resistance. Several enhancer elements have been shown to regulate mouse Nodal expression and studies have delineated mechanisms by which mRNA splicing and translation of NODAL homologues are regulated in model organisms. However, little is known regarding the co-transcriptional and post-transcriptional processing of human NODAL. Herein, we describe hitherto unreported RNAs which are transcribed from the NODAL locus, including an antisense transcript, a circular transcript, and multiple splice variants. These transcripts demonstrate the complexity of NODAL expression and highlight the need to consider each NODAL variant when attempting to quantify or target this morphogen.

molecular biology↗

bioRxiv2018

Genomic copy-number loss is rescued by self-limiting production of DNA circles

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC production is proportional to repeat loss and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Lastly, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

molecular biology↗

bioRxiv2018

Caenorhabditis elegans heterochromatin factor SET-32 plays an essential role in transgenerational establishment of nuclear RNAi-mediated epigenetic silencing

Epigenetic inheritance contributes fundamentally to transgenerational physiology and fitness. Mechanistic understanding of RNA-mediated chromatin modification and transgenerational epigenetic inheritance, which in C. elegans can be triggered by exogenous double-stranded RNA (exo-dsRNA) or facilitated by endogenous small interfering RNAs (endo-siRNAs), has mainly been limited to the post-initiation phases of silencing. Indeed, the dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here we found that the onset of exo-dsRNA-induced nuclear RNAi is a transgenerational process, and that establishment requires SET-32, one of the three putative histone methyltransferases (HMTs) that are required for H3K9me3 deposition at the nuclear RNAi targets. We also performed multigenerational whole-genome analyses to examine the establishment of silencing at endogenous targets of germline nuclear RNAi. The nuclear Argonaute protein HRDE-1 is essential for the maintenance of nuclear RNAi. Repairing a loss-of-function mutation in hrde-1 by CRISPR restored the silencing of endogenous targets in animals carrying wild type set-32. However, for numerous endogenous targets, repairing the hrde-1 mutation in a set-32;hrde-1 double mutant failed to restore their silencing states in up to 20 generations after the hrde-1 repair, using a similar genome editing approach. We found that despite a prominent role in the establishment of silencing, however, set-32 is completely dispensable for the maintenance of silencing once HRDE-1-dependent gene repression is established. Our study indicates that: 1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements; and 2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process. In addition, our study reveals a novel paradigm in which a heterochromatin factor primarily functions to promote the establishment of transgenerational silencing, expanding mechanistic understanding of the well-recognized role of heterochromatin in epigenetic maintenance.

molecular biology↗

bioRxiv2018

Cotranslational folding of a pentarepeat β-helix protein

It is becoming increasingly clear that many proteins start to fold cotranslationally, before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, i.e., proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a {beta}-helix pentarepeat protein from the human pathogen Clostridium botulinum - a homolog of the Fluoroquinolone Resistance Protein MfpA - using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight {beta}-helix coils in the protein produces enough force to release ribosome stalling, and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo, and may reflect the minimal size of a stable {beta}-helix since it is structurally homologous to the smallest known {beta}-helix protein, a four-coil protein that is stable in solution.

molecular biology↗

bioRxiv2018

Nucleosome turnover is sufficient to establish varied histone methylation states

Transcription-dependent methylation of histone H3 at lysine 79 (H3K79) is evolutionarily conserved from yeast to mammals, critical for normal development and frequently deregulated by genetic recombination in Mixed Lineage Leukemia. Although this histone modification is associated with gene activity, little is known about the cellular mechanisms of H3K79 methylation regulation. Because no H3K79 demethylase has been discovered, the mechanism of its removal remains unclear. Utilizing chemical-induced-proximity to control histone methylation in vivo we show that the dynamics of methylation state (mono, di, tri-methylation) is genome-context specific. Further, Monte Carlo simulations coupling systems of kinetic reactions with histone turnover rates, suggest that nucleo-some turnover is sufficient to establish varied genome-wide methylation states without active demethylation.

molecular biology↗

bioRxiv2018

Cryo-EM structure of Escherichia coli σ70 RNAP and promoter DNA complex revealed a role of σ nonconserved region during the open complex formation

First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary {sigma}70 factor form the RNAP holoenzyme to express housekeeping genes. The {sigma}70 contains a large insertion at between the conserved regions 1.2 and 2.1, the {sigma} non-conserved region ({sigma}NCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP {sigma}70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 [A] resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between R157 residue in the {sigma}NCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography due to restraint of crystal packing effect. Disruption of the {sigma}NCR and DNA interaction by the amino acid substitution (R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the {sigma}NCR and DNA interaction is conserved in proteobacteria and RNAP in other bacteria replace its role with a transcription factor.

molecular biology↗

bioRxiv2018

Rbfox splicing factors maintain skeletal muscle mass by regulating calpain3 and proteostasis

Alternative splicing promotes proteomic diversity important for cellular differentiation and cell fate determination. Here, we show that deletion of the highly conserved Rbfox1 and Rbfox2 alternative splicing regulators in adult mouse skeletal muscle causes rapid, severe loss of muscle mass. Homeostasis of skeletal muscle tissue requires a dynamic balance between protein synthesis and degradation (proteostasis) but the mechanisms that regulate this balance are not well understood. Rbfox deletion did not cause reduced global protein synthesis, but resulted in reduced autophagy flux and altered splicing of hundreds of transcripts including Capn3, which produced an active form of calpain3 protease. The results indicate Rbfox proteins regulate proteostasis in skeletal muscle tissue by control of calpain and autophagy-lysosome pathways.\n\nHighlightsO_LIProteostasis in adult skeletal muscle is post-transcriptionally regulated, in part by alternative splicing via Rbfox1/2\nC_LIO_LIRbfox1/2 regulate hundreds of targets in skeletal muscle, including Calpn3, to maintain muscle mass in adult mice\nC_LIO_LIAutophagy flux is markedly decreased in muscle lacking Rbfox1/2\nC_LIO_LIAs for neurons, altered proteostasis is detrimental to adult muscle\nC_LI

molecular biology↗

bioRxiv2018

The plasma miR-122 basal levels respond to circulating catecholamine in rats

miR-122 in circulation is a promising non-invasive biomarker as a replacement or supplement of current serum biomarkers for liver injuries. But the concept was questioned by recent studies, mainly due to its release from hepatocytes in absence of overt cellular injuries. In this study, we reported that the hepatic metabolism of circulating catecholamines resulted in the release of hepatocyte-specific miR-122. Acute stress-induced hepatocellular deformation was histopathologically different from drug-induced liver injury with significant increases of plasma miR-122 levels. The basal levels of human plasma miR-122 could be significantly altered by emotional responses. Interday variances of plasma miR-122 measurements were reduced effectively by stress-relief measures. The metabolism of basal circulating norepinephrine and epinephrine in liver might contribute to the basal levels of plasma miRNAs expressed in hepatocytes.

molecular biology↗

bioRxiv2018

Titer estimation for quality control (TEQC) method: a practical approach for optimal production of protein complexes using the baculovirus expression vector system

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain the protein or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the initial infectivity was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

molecular biology↗

bioRxiv2018

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1kb) retained introns than they are in small retained introns or in non-retained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

molecular biology↗

bioRxiv2018

Early candidate urine biomarkers for detecting Alzheimer’s disease before beta amyloid plaque deposition in an APP (swe)/PSEN1dE9 transgenic mouse model

Alzheimers disease (AD) is an incurable age-associated neurodegenerative disorder that is characterized by irreversible progressive cognitive deficits and extensive brain damage. The identification of candidate biomarkers before beta amyloid plaque deposition occurs is therefore of great importance for the early intervention of AD. Urine, which is not regulated by homeostatic mechanisms, theoretically accumulates changes associated with AD earlier than cerebrospinal fluid and blood. In this study, an APP (swe)/PSEN1dE9 transgenic mouse model was used to identify candidate biomarkers for early AD. Urine samples were collected from 4-, 6-, and 8-month-old transgenic mouse models, and the urinary proteomes were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The levels of 33 proteins differed significantly between wild-type and 4-month-old mice, which had not started to deposit beta amyloid plaque. Among these proteins, 16 have been associated with the mechanisms of AD, while 9 have been suggested as AD biomarkers. Our results indicated that urine proteins enable detecting AD before beta amyloid plaque deposition, which may present an opportunity for intervention.

molecular biology↗