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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Let-7i-5p regulation of cell morphology and migration through distinct signaling pathways in normal and pathogenic fibroblasts from urethra

Pelvic fracture urethral distraction defects (PFUDD) is a common disease that could severely affect patients life quality, yet little is known about the molecular mechanism associated with pathogenic fibrosis in PFUDD. In this study, we found that let-7i-5p could regulate different cellular events in normal and pathogenic fibroblasts through three distinct signaling pathways. Interestingly, those regulations are compromised during the translation from mRNA to protein, and partially based on pathogenic status of the fibroblasts. By analyzing the molecular mechanism associated with its function, we conclude that let-7i-5p plays an essential role in regulating cell shape and tissue elasticity, cell migration, cell morphology and cytoskeleton, and could serve as a potential target for clinical treatment of urethral stricture patients.

molecular biology↗

bioRxiv2018

Condensin controls cellular RNA levels through the accurate segregation of chromosomes instead of directly regulating transcription

Condensins are genome organisers that shape chromosomes and promote their accurate transmission. Several studies have also implicated condensins in gene expression, although the mechanisms have remained enigmatic. Here, we report on the role of condensin in gene expression in fission and budding yeasts. In contrast to previous studies, we provide compelling evidence that condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis. We further show that the changes in gene expression in post-mitotic fission yeast cells that result from condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite condensin inactivation. Thus, chromosome instability, rather than a direct role of condensin in the transcription process, changes gene expression. This knowledge challenges the concept of gene regulation by canonical condensin complexes.

molecular biology↗

bioRxiv2018

Development and Validation of a Method for Human Papillomavirus Genotyping Based on Molecular Beacon Probes

We describe a new assay system for the genotyping of human papillomavirus (HPV) based on linear-after-the-exponential-PCR and melting curve analysis. This system can detect and identify the 23 most common HPV strains (types 6, 11, 16, 18, 31, 33, 35, 39,42, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 81, 82, and 83) in two sealed reaction tubes within 2 h. The sensitivity and specificity of this new system was validated using cloned HPV DNA and clinical samples. The detection limit was 5-500 copies/reaction depending on the genotype, and no cross-reactivity was observed with any other low-risk HPV or pathogen that was commonly found in the female genital tract. When compared with the HPV GenoArray test kit, the testing of 1104 clinical samples produced a good overall agreement between the two methods of 98.37% (95% CI: 97.44%-98.97%) and a kappa value of 0.954. Thus, this new HPV genotyping assay system represents a simple, rapid, universally applicable, sensitive, and highly specific detection methodology that should be useful for cervical lesion screening and, thus, is potentially of great value in future clinical applications.

molecular biology↗

bioRxiv2018

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

molecular biology↗

bioRxiv2018

pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli

N-terminal acetylation is one of the most common post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular -synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely N-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely N-acetylate our model substrate -synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.

molecular biology↗

bioRxiv2018

Multiplexed Primer Extension Sequencing Enables High Precision Detection of Rare Splice Isoforms

Targeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. Here we present a novel approach for targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. We demonstrate this approach by targeting hundreds to thousands of pre-mRNA splice junctions, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

molecular biology↗

bioRxiv2018

UPA-Seq: Prediction of Functional LncRNAs Using Differential Sensitivity to UV Crosslinking

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-Seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-Seq). As expected, the numbers of UPA-Seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-Seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

molecular biology↗

bioRxiv2018

Genetic tools weed out misconceptions of strain reliability in Cannabis sativa: Implications for a budding industry

Cannabis sativa is listed as a Schedule I substance by the United States Drug Enforcement Agency and has been federally illegal in the United States since 1937. However, the majority of states in the United States, as well as several countries, now have various levels of legal Cannabis. Products are labeled with identifying strain names but there is no official mechanism to register Cannabis strains, therefore the potential exists for incorrect identification or labeling. This study uses genetic analyses to investigate strain reliability from the consumer point of view. Ten microsatellite regions were used to examine samples from strains obtained from dispensaries in three states. Samples were examined for genetic similarity within strains, and also a possible genetic distinction between Sativa, Indica, or Hybrid types. The analyses revealed genetic inconsistencies within strains. Additionally, although there was strong statistical support dividing the samples into two genetic groups, the groups did not correspond to commonly reported Sativa/Hybrid/Indica types. Genetic differences have the potential to lead to phenotypic differences and unexpected effects, which could be surprising for the recreational user, but have more serious implications for patients relying on strains that alleviate specific medical symptoms.

molecular biology↗

bioRxiv2018

Imaging of single mRNA translation repression reveals diverse interactions with mRNP granules

During cellular stress mRNAs exit translation and accumulate in stress granules and P-bodies, although the dynamics of these interactions remain unclear. We imaged in real-time single mRNAs, their translational output, and mRNA-granule interactions during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionality between them. While translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable associations that rigidly immobilize the mRNA within the granule. Imaging thousands of individual mRNA-granule interactions showed the probability of stable association increases with both mRNA length and granule size. Therefore, the recruitment of mRNAs to RNP granules involves both highly dynamic and stable interactions, influenced by several parameters, demonstrating a new layer of complexity in mRNA regulation during stress.\n\nOne Sentence SummarymRNAs interact with stress granules and P-bodies in stable and dynamic manners influenced by ribosome association, mRNA length, and granule size.

molecular biology↗

bioRxiv2018

Effect of automation on the accuracy of alchemical free energy calculation protocols

Hit-to-lead virtual screening frequently relies on a cascade of computational methods that starts with rapid calculations applied to a large number of compounds and ends with more expensive computations restricted to a subset of compounds that passed initial filters. This work focuses on set up protocols for alchemical free energy (AFE) scoring in the context of a Docking - MM/PBSA - AFE cascade. A dataset of 15 congeneric inhibitors of the ACK1 protein was used to evaluate the performance of AFE set up protocols that varied in the steps taken to prepare input files (using previously docked and best scored poses, manual selection of poses, manual placement of binding site water molecules). The main finding is that use of knowledge derived from X-ray structures to model binding modes, together with the manual placement of a bridging water molecule, improves the R2 from 0.45 {+/-} 0.06 to 0.76 {+/-} 0.02 and decreases the mean unsigned error from 2.11 {+/-} 0.08 to 1.24 {+/-} 0.04 kcal mol-1. By contrast a brute force automated protocol that increased the sampling time ten-fold lead to little improvements in accuracy. Besides, it is shown that for the present dataset hysteresis can be used to flag poses that need further attention even without prior knowledge of experimental binding affinities.

molecular biology↗

bioRxiv2018

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

molecular biology↗

bioRxiv2018

xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

molecular biology↗

bioRxiv2018

Depolymerized lamins link nuclear envelope breakdown to mitotic transcriptional quiescence

The nuclear envelope, a defining feature of eukaryotic cells, restricts DNA-dependent processes including gene transcription to the nucleus. The nuclear lamina is an integral component of the animal nuclear envelope, composed of polymers of nuclear lamin proteins1,2. Upon mitosis, the nuclear lamina disassembles, the nuclear envelope breaks down, and transcription becomes quiescent3,4. We report here a direct molecular link between nuclear lamina disassembly and mitotic transcriptional quiescence. We found that, at the G2 cell-cycle phase immediately preceding mitosis, nuclear lamin A/C (LMNA) became phosphorylated at Ser22 and depolymerized from the nuclear lamina. Depolymerized LMNA accumulated in the nuclear interior and physically associated with active cis-regulatory elements genome-wide. Depolymerized LMNA-associated sites were overrepresented near genes repressed by LMNA, suggesting that depolymerized LMNA participates in transcriptional repression at G2. Consistently, depolymerized LMNA-target genes underwent a steep expression decline from S to G2/M. Furthermore, LMNA deletion caused inappropriate RNA Polymerase II (Pol II) accumulation downstream of Pol II pause sites at promoters and enhancers genome-wide, leading to inappropriate and excessive transcriptional elongation. A subset of depolymerized LMNA-target genes were upregulated in fibroblasts of patients with Hutchinson-Gilford progeria, a premature aging disorder caused by LMNA mutations5, raising the possibility that defects in depolymerized LMNA-mediated mitotic transcriptional quiescence contribute to progeria pathogenesis. These observations support a model in which depolymerized LMNA targets active regulatory elements to promote RNA Pol II pausing preceding mitosis, coupling nuclear envelope breakdown to mitotic transcriptional quiescence.

molecular biology↗

bioRxiv2018

The RASSF6 tumor suppressor protein regulates apoptosis and cell cycle progression via Retinoblastoma protein.

RASSF6 is a member of the tumor suppressor Ras-association domain family (RASSF) proteins. RASSF6 is frequently suppressed in human cancers and its low expression is associated with poor prognosis. RASSF6 regulates cell cycle arrest and apoptosis and plays a tumor suppressor role. Mechanistically, RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. However, RASSF6 also induces cell cycle arrest and apoptosis in the p53-negative background, which implies that the tumor suppressor function of RASSF6 does not depend solely on p53. In this study, we have revealed that RASSF6 mediates cell cycle arrest and apoptosis via pRb. RASSF6 enhances the interaction between pRb and protein phosphatase. RASSF6 also enhances P16INK4A and P14ARF expression through suppressing BMI1. In this way, RASSF6 increases unphosphorylated pRb and augments the interaction between pRb and E2F1. Moreover, RASSF6 induces TP73-target genes via pRb and E2F1 in the p53-negative background. Finally, we confirmed that RASSF6 depletion induces polypoid cells in p53-negative HCT116 cells. In conclusion, RASSF6 behaves as a tumor suppressor in cancers with the loss-of-function of p53, and pRb is implicated in this function of RASSF6.

molecular biology↗

bioRxiv2018

Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.\n\nAuthor summaryThe antiretroviral drugs (ARVs) such as LP or SQ that inhibit viral proteases reduce the rate of multiplication of the malaria parasites. The mode of action of these drugs against the parasites is however poorly understood. The proteases are among the enzymes that play essential roles in Plasmodium parasites. We sought to investigate the possible mode of action of these drugs by generating mutant parasites lacking specific aspartyl proteases namely PM4, PM7, PM8 or Ddi1 and then evaluate the susceptibility of the mutants to LP and SQ. We successfully generated parasites lacking either PM4, PM7 or PM8 but Ddi1 gene was refractory to deletion. From our data, we demonstrate that, unlike PM7 and PM8, the PM4 and Ddi1 are essential enzymes for asexual blood stage parasite fitness and survival and that the PM4 might be a target for the viral protease inhibitors in reducing parasite growth and multiplication. Further experiments using molecular docking tools show that LP or SQ have a high binding affinity for the Plasmodium aspartyl proteases.

molecular biology↗

bioRxiv2018

Comparative evaluation of the GeeniusTM HIV 1/2 Confirmatory Assay and the HIV-1 and HIV-2 Western blots in the Japanese population

Accurate diagnosis of earlier HIV infection is essential for treatment and prevention. Currently, confirmation tests of HIV infection in Japan are performed using Western blot (WB), but WB has several limitations including low sensitivity and cross-reactivity between HIV-1 and HIV-2 antibodies. To address these problems, a new HIV testing algorithm and a more reliable confirmation and HIV-1/2 differentiation assay are required. The Bio-Rad Geenius HIV-1/2 Confirmatory Assay (Geenius) has recently been approved and recommended for use in the revised guidelines for diagnosis of HIV infection by the Center for Disease Control and Prevention (USA). We made comprehensive comparison of the performance of Geenius and the Bio-Rad NEW LAV BLOT 1 and 2 (NLB 1 and 2) which are WB kits for HIV-1 and HIV-2, respectively, to examine if Geenius is a suitable alternative to these WB assays which are now being used in HIV testing in Japan. A total of 166 HIV-1 positive samples (146 from patients with established HIV-1 infection and 20 from patients with acute infection), five HIV-1 seroconversion panels containing 21 samples and 30 HIV-2 positive samples were used. In addition, a total of 140 HIV negative samples containing 10 false-positives on screening tests were examined. The sensitivity of Geenius and NLB 1 for HIV-1 positive samples was 99.3% and 98.6%, respectively. Geenius provided more positive results in the samples from acute infections and detected positivity 0 to 32 days earlier in seroconversion panels than NLB 1. NLB 2 gave positive results in 12.3% of HIV-1 positive samples. The sensitivity of both Geenius and NLB 2 for HIV-2 positive samples was 100%. The specificity of Geenius, NLB 1 and NLB 2 was 98.5%, 81.5% and 90.0%, respectively. Geenius is an attractive alternative to WB for confirmation and differentiation of HIV-1 and HIV-2 infections. The adaptation of Geenius to the HIV testing algorithm may be advantageous for rapid diagnosis and the reduction of testing costs.

molecular biology↗

bioRxiv2018

Epidemiologal investigation of an Acinetobacter baumannii outbreak using Core Genome Multilocus Sequence Typing

Carbapenem-resistant (CR) Acinetobacter baumannii is a serious nosocomial pathogen able to cause a variety of serious, often life-threatening infections and outbreaks. We aimed to investigate the molecular epidemiology of clinical isolates of CR-A. baumannii from an outbreak occurred in the intensive care unit (ICU) of our hospital.\n\nFrom January to April 2017, 13 CR-A. baumannii isolates were collected at the \"L. Spallanzani\" hospital, Rome, Italy; typing was performed by repetitive extragenetic palindromic-based (rep)- PCR-based DiversiLab system and WGS data were used for in silico analysis of traditional MLST types, for identifying resistance genes and for core genome multi locus sequence typing (cgMLST) analysis. Epidemiological data were obtained from hospital records.\n\nStrains were cultured from 7 patients treated in the ICU of our hospital; all isolates showed a multi-drug resistant (MDR) profile, carrying the blaOXA-23 carbapenemase. Typing performed by rep-PCR and MLST showed that the isolates clustered into one group while the cgMLST approach, which uses 2690 gene targets to characterize the gene-by-gene allelic profile of A. baumannii, highlighted the presence of two cluster types. These results allowed us to identify two patients who entered the ICU already colonized by two different strains of CR-A. baumannii; we hypothesize that these two patients could be the source of two separate transmission chains.\n\nOur results show that whole-genome-DNA sequencing by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.

molecular biology↗

bioRxiv2018

IL-11 is a therapeutic target in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) remains a progressive disease despite best medical management. We previously identified IL-11 as a critical factor for cardiovascular fibrosis and examine here its role in pulmonary fibrosis. IL-11 is consistently upregulated in IPF genomic datasets, which we confirmed by histology. Pulmonary fibroblasts stimulated with IL-11 transform into invasive myofibroblasts whereas fibroblasts from Il11ra deleted mice did not respond to pro-fibrotic stimuli. In the mouse, injection of recombinant Il-11 or fibroblast-specific expression of Il-11 caused pulmonary fibrosis. We then generated a neutralising IL-11 binding antibody that blocks lung fibroblast activation across species. In a mouse model of IPF, anti-IL-11 therapy attenuated lung fibrosis and specifically blocked Erk activation. These data prioritise IL-11 as an accessible drug target in IPF.\n\nOne Sentence SummaryNon-canonical IL-11 signalling is a central hallmark of idiopathic pulmonary fibrosis and represents a novel target for antibody therapies.

molecular biology↗