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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

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↗

bioRxiv2018

Loss of SDHB reprograms energy metabolisms and inhibits high fat diet induced metabolic syndromes

Mitochondrial respiratory complex II utilizes succinate, key substrate of the Krebs cycle, for oxidative phosphorylation, which is essential for glucose metabolism. Mutations of complex II cause cancers and mitochondrial diseases, raising a critical question of the (patho-)physiological functions. To address the fundamental role of complex II in systemic energy metabolism, we specifically knockout SDHB in mice liver, a key complex II subunit that tethers the catalytic SDHA subunit and transfers the electrons to ubiquinone, and found that SHDB deficiency abolishes the assembly of complex II without affecting other respiration complexes while largely retaining SDHA stability. SHDB ablation reprograms energy metabolism and hyperactivates the glycolysis, Krebs cycle and {beta}-oxidation pathways, leading to catastrophic energy deficit and early death. Strikingly, sucrose supplementation or high fat diet resumes both glucose and lipid metabolism and prevent early death. Also, SDHB deficient mice are completely resistant to high fat diet induced obesity. Our findings reveal that the unanticipated role of complex II orchestrating both lipid and glucose metabolisms, and suggest that SDHB is an ideal therapeutic target for combating obesity.

molecular biology↗

bioRxiv2018

Apolipoprotein L1 Dynamics in Human Parietal Epithelial Cell Molecular Phenotype Kinetics

Human Parietal Epithelial cells (PECs) are considered as a source of progenitor cells to sustain podocyte (PD) homeostasis. We hypothesized that the absence of apolipoprotein (APO) L1 favors the PEC phenotype and that induction of APOL1 transitions to PD renewal. During PECs transition, APOL1 expression coincided with the expression of PD markers (PEC transition) along with down regulation of miR193a. The induction of APOL1 down regulated miR193a and induced PD markers in PECs/HEKs; whereas, the APOL1-silencing in transited (Tr)-PECs/HepG2s up regulated miR193a expression suggesting a reciprocally linked feedback loop relationship between APOL1 and miR193a. HIV, IFN-y, and vitamin D receptor agonist (VDA) induced APOL1 expression and PEC transition markers but down regulated miR193a in PECs/HEKs. Glomeruli in HIV patients and HIV: APOL1 transgenic mice displayed foci of PECs expressing synaptopodin, a PEC transition marker. Since APOL1 silencing in PECs partially attenuated HIV-, VDA-, and IFN-y-induced PECs transition, this would suggest that APOL1 is an important functional constituent of APOL1-miR193a axis.

molecular biology↗

bioRxiv2018

Template switching causes artificial junction formation and false identification of circular RNAs

Hundreds of thousands of putative circular RNAs have been identified through deep sequencing and bioinformatic analyses. However, the circularity of these putative RNA circles has not been experimentally validated due to limited methodologies currently available. We reported here that the template-switching capability of commonly used reverse transcriptases (e.g., SuperScript II) leads to the formation of artificial junction sequences, and consequently misclassification of large linear RNAs as RNA circles. Use of reverse transcriptases without terminal transferase activity (e.g., MonsterScript) for cDNA synthesis is critical for the identification of physiological circular RNAs. We also report two methods, MonsterScript junction PCR and high-resolution melting curve analyses, which can reliably distinguish circular RNAs from their linear forms and thus, can be used to discover and validate true circular RNAs.\n\nSignificance StatementThe vast majority of circular RNAs were identified through computational detection of junction sequences in the deep sequencing reads because these unique fusion sequences represent back-splicing events. We found that artificial junction sequences could be formed through template switching (TS) when MMLV-derived reverse transcriptases, e.g., SuperScript II, are used to synthesize cDNAs. Thus, many of the reported circular RNAs may not be RNA circles, but rather experimental artifacts. Fake circular RNAs can be avoided by using reverse transcriptases without terminal transferase activity (e.g., MonsterScript) for cDNA synthesis. We developed two novel methods, MonsterScript junction PCR and high-resolution melting curve analyses, for distinguishing circular RNAs from their linear form.

molecular biology↗

bioRxiv2018

Herpes ICP8 protein stimulates homologous recombination in human cells

Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red SynExo homologous recombination proteins Beta in Escherichia coli has approached 100% efficiency. While highly efficient in E. coli, recombineering using the Red SynExo in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus Type 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a \"humanized\" version of Beta protein from phage {lambda}. ICP8 significantly enhanced gene targeting rates in HEK 293 T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.\n\nSIGNIFICANCEGenome modification by recombineering using SynExo viral recombination proteins has transformed functional genomic analysis in bacteria. Single-stranded DNA (ssDNA) recombineering approaches 100% efficiency in E. coli using Beta protein from bacteriophage lambda, but recombineering has not been extended to eukaryotic genomes. Efficient recombineering requires SynExos that co-evolved with a viral host, however SynExos are common to viruses infecting a variety of organisms, including humans. The ICP8 protein of Human Herpes virus Type 1 is a SynExo protein similar to Beta. In this pioneering study, Herpes ICP8 stimulated gene targeting in a human genome by homologous recombination while the bacterial virus Beta protein inhibited recombination in human cells. This is the first demonstration of host-specific recombineering in human cells using a human viral SynExo protein.

molecular biology↗

bioRxiv2018

First Efficient Transfection in Choanoflagellates using Cell-Penetrating Peptides

Only recently, based on phylogenetic studies choanoflagellates have been confirmed to form the sister group to metazoan. The mechanisms and genes behind the step from single to multicellular organisation and as a consequence the evolution of metazoan multicellularity could not be verified yet, as no reliable and efficient method for transfection of choanoflagellates was available. Here we present cell-penetrating peptides (CPPs) as an alternative to conventional transfection methods. In a series of experiments with the choanoflagellate Diaphanoeca grandis we proof for the first time that the use of CPPs is a reliable and highly efficient method for the transfection of choanoflagellates. We were able to silence the silicon transporter gene (SIT) by siRNA, and hence, to suppress the lorica (characteristic siliceous basket) formation. High gene silencing efficiency was determined and measured by light microscope and RT-qPCR. In addition, only low cytotoxic effects of CPP were detected. Our new method allows the reliable and efficient transfection of choanoflagellates, finally enabling us to verify the function of genes, thought to be involved in cell adhesion or cell signaling by silencing them via siRNA. This is a step stone for the research on the origin of multicellularity in metazoans.

molecular biology↗

bioRxiv2018

Prediction of the secundary structure at the tRNASer (UCN) of Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis is the main vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in America and Colombia. Taxonomically belongs to the subgenus Lutzomyia, which includes other vector species that exhibit high morphological similarity to the female species difficult to identify vectors in leishmaniasis foci and suggesting the search for molecular markers that facilitate this task, further researchs with mitochondrial genes, chromosome banding, reproductive isolation and pheromones evidence the existence of species complex. The aim of this study was to predict the secondary structure of mitochondrial transfer RNA serine (tRNASer) for UCN codon of Lutzomyia longipalpis as molecular marker for identify of this species. Sequences recorded in Genbank of L. longipalpis sequences were aligned with tRNA's from previously described species and then tRNASer secondary structure was inferred by software tRNAscan-SE 1.21. The length of tRNASer was 67 base pairs (bp). Two haplotypes were detected in the five sequences analyzed. The L. longipalpis tRNASer showed 7 intrachain pairing in the acceptor arm, 3 in the DHU arm, 4 in the anticodon arm and 5 in the T{psi}C. The size of the loops corresponded to 5 nucleotides in the DHU, 7 in the anticodon, 4 in the variable and 7 in the T{psi}C. L. longipalpis is distinguished from other species at subgenera Lutzomyia by the secondary structure and substitutions inferred tRNASer evidenced in the primary sequence.

molecular biology↗

bioRxiv2018

Regulation of ATM and ATR by SMARCAL1 and BRG1

The G2/M checkpoint is activated on DNA damage by the ATM and ATR kinases that are regulated by post-translational modifications. In this paper, the transcriptional co-regulation of ATM and ATR by SMARCAL1 and BRG1, both members of the ATP-dependent chromatin remodeling protein family, is described. SMARCAL1 and BRG1 co-localize on the promoters of ATM and ATR; downregulation of SMARCAL1/BRG1 results in transcriptional repression of ATM/ATR and therefore, overriding of the G2/M checkpoint leading to mitotic abnormalities. On doxorubicin-induced DNA damage, SMARCAL1 and BRG1 are upregulated and in turn, upregulate the expression of ATM/ATR.\n\nPhosphorylation of ATM/ATR is needed for the transcriptional upregulation of SMARCAL1 and BRG1, and therefore, of ATM and ATR on DNA damage. The regulation of ATM/ATR is rendered non-functional if SMARCAL1 and/or BRG1 are absent or if the two proteins are mutated such that they are unable to hydrolyze ATP, as in for example in Schimke Immuno-Osseous Dysplasia and Coffin-Siris Syndrome. Thus, an intricate transcriptional regulation of DNA damage response genes mediated by SMARCAL1 and BRG1 is present in mammalian cells.

molecular biology↗

bioRxiv2018

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the proteins binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

molecular biology↗

bioRxiv2018

Functional characterization of sensory neuron membrane proteins (SNMPs)

Sensory neuron membrane proteins (SNMPs) play a critical role in the insect olfactory system but there is a deficit of functional studies beyond Drosophila. Here, we provide functional characterisation of insect SNMPs through the use of bioinformatics, genome curation, transcriptome data analysis, phylogeny, expression profiling, and RNAi gene knockdown techniques. We curated 81 genes from 35 insect species and identified a novel lepidopteran SNMP gene family, SNMP3. Phylogenetic analysis shows that lepidopteran SNMP3, but not the previously annotated lepidopteran SNMP2, is the true homologue of the dipteran SNMP2. Digital expression, microarray and qPCR analyses show that the lepidopteran SNMP1 is specifically expressed in adult antennae. SNMP2 is widely expressed in multiple tissues while SNMP3 is specifically expressed in the larval midgut. Microarray analysis suggest SNMP3 may be involved in the silkworm immunity response to virus and bacterial infections. We functionally characterised SNMP1 in the silkworm using RNAi and behavioural assays. Our results suggested that Bombyx mori SNMP1 is a functional orthologue of the Drosophila melanogaster SNMP1 and plays a critical role in pheromone detection. Split-ubiquitin yeast hybridization study shows that BmorSNMP1 has a protein-protein interaction with the BmorOR1 pheromone receptor, and the BmorOrco co-receptor. Concluding, we propose a novel molecular model in which BmorOrco, BmorSNMP1 and BmorOR1 form a heteromer in the detection of the silkworm sex pheromone bombykol.

molecular biology↗

bioRxiv2018

Environmentally enriched pigs have transcriptional profiles consistent with neuroprotective effects and microglial depletion

Environmental enrichment (EE) is widely used to study the effects of external factors on brain development, function and health in rodent models, but very little is known of the effects of EE on the brain in a large animal model such as the pig. Twenty-four young pigs (aged 5 weeks at start of study, 1:1 male: female ratio) were housed in environmentally enriched (EE) pens and provided with additional enrichment stimulation (a bag filled with straw) once daily. Litter, weight and sex matched controls n= (24) were housed in barren (B) conditions. Behaviour was recorded on alternate days from study day 10. After 21 days, RNA-sequencing of the frontal cortex of male piglets culled one hour after the enrichment stimulation, but not those at 4 hours after stimulation, showed upregulation of genes involved in neuronal activity and synaptic plasticity in the EE compared to the B condition. This result is mirrored in the behavioural response to the stimulation which showed a peak in activity around the 1 hour time-point. By contrast, EE piglets displayed a signature consistent with a relative decrease in microglial activity compared to those in the B condition. These results confirm those from rodents, suggesting that EE may also confer neuronal health benefits in large mammal models, through a potential relative reduction in neuroinflammatory process and increase in neuroprotection driven by an enrichment-induced increase in behavioural activity.

molecular biology↗

bioRxiv2018

RAD51 Enhances Zygotic Interhomolog Repair

Recent advances in genome editing have greatly improved knock-in (KI) efficiency1-9. Searching for factors to further improve KI efficiency for therapeutic use and generation of non-human primate (NHP) models, we found that the strand exchange protein RAD51 can significantly increase homozygous KI using CRISPR/Cas9 in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is well-described in the context of meiosis10, but only occurs at low frequencies in mitotic cells11,12 and its existence in zygotes is controversial. Using a variety of approaches, we provide evidence for an endogenous IHR mechanism in zygotes that can be enhanced by RAD51. We show that this process can be harnessed for generating homozygous KI animals from wildtype zygotes based on exogenous donors and for converting heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we elucidate additional factors that contribute to zygotic IHR and identify a RAD51 mutant capable of insertion-deletion (indel)-free stimulation of IHR. Thus, our study provides conclusive evidence for the existence of zygotic IHR and demonstrates methods to enhance IHR for potential use in gene drives, gene therapy, and biotechnology.

molecular biology↗

bioRxiv2018

miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential retroviral restriction factor in macrophages

The goal of this study was to characterize extracellular vesicles (EVs) and miRNAs of primate cervicovaginal lavage (CVL) during the menstrual cycle and simian immunodeficiency virus (SIV) infection, and to determine if differentially regulated CVL miRNAs might influence retrovirus replication. CVL and peripheral blood were collected from SIV-infected and uninfected macaques. EVs were enriched by stepped ultracentrifugation and characterized thoroughly. miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform and validated by single qPCR assays. Hormone cycling was abnormal in infected subjects, but EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs were found to vary during the menstrual cycle or SIV infection. Among them was miR-186-5p, which was depleted in retroviral infection. In experiments with infected macrophages in vitro, this miRNA inhibited HIV replication. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract.

molecular biology↗

bioRxiv2018

Heat shock induces the depletion of Oct4 in mouse blastocysts and stem cells

Temperature is an important microenvironmental factor that functions epigenetically in normal embryonic development. However, the effect of heat shock in the stem cells is not fully understood. Oct4 is a tightly regulated master regulator of pluripotency maintenance in stem cells and during early embryonic development. We report here that Oct4 protein level was significantly reduced under heat shock in mouse blastocysts and embryonic stem cells. The reduction in Oct4 in the mouse embryonic stem cells under heat shock was mediated by a ubiquitin-proteasome pathway that was dependent on the activity of death- associated protein kinase 1 (Dapk1) to phosphorylate its substrate, Pin1. Our results imply that the depletion of Oct4 via brief heat shock, such as a high fever, during early pregnancy might severely impair the growth of the mammalian embryo or even cause its death.

molecular biology↗

bioRxiv2018

A Novel QconCAT-Based Proteomics Method for Determining Allele-Specific Protein Expression (ASPE): a New Approach to Identify Cis-acting Genetic Variants

Measuring allele-specific expression (ASE) is a powerful approach for identifying cis-regulatory genetic variants. Here we developed a novel targeted proteomics method for quantification of allele-specific protein expression (ASPE) based on scheduled high resolution multiple reaction monitoring (sMRM-HR) with a heavy stable isotope-labeled quantitative concatamer (QconCAT) internal protein standard. This strategy was applied to the determination of the ASPE of UGT2B15 in human livers using the common UGT2B15 nonsynonymous variant rs1902023 (i.e. Y85D) as the marker to differentiate expressions from the two alleles. The QconCAT standard contains both the wild type tryptic peptide and the Y85D mutant peptide at a ratio of 1:1 to ensure accurate measurement of the ASPE of UGT2B15. The results from 18 UGT2B15 Y85D heterozygotes revealed that the ratios between wild type Y allele and mutant D allele varied from 0.60 to 1.46, indicating the presence of cis-regulatory variants. In addition, we observed no significant correlations between the ASPE and mRNA ASE of UGT2B15, suggesting the involvement of different cis-acting variants in regulating the transcription and translation processes of the gene. This novel ASPE approach provides a powerful tool for capturing cis-genetic variants involved in post-transcription processes, an important yet understudied area of research.

molecular biology↗