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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2015

Connecting small RNAs and Aging

Website SummarySmall RNAs are important gene regulators of stress response, aging, and many other things. More analysis needs to be done in order to gain a better understanding of these molecules to find connections between small RNAs and important things like aging.\n\nSummarySmall RNAs are a diverse population of gene regulators, but their role in the cell is not fully characterized. Bioinformatics was used to prove their connection with aging and expand current knowledge for these molecules.\n\nAbstractSmall RNAs have a wide range of functions and recent studies have found connections between these molecules and aging pathways. However, the process to systematically characterize this relationship is slow. Prediction tools can be used to expedite this process by finding new genes and pathways that cross talk with each other. Using phylogenetic and systems analysis, connections between small RNAs and aging were proven and new genes that may be related to aging were identified. This type of analysis can be applied to many different pathways in order to fully characterize the role of small RNAs.

Molecular Biology↗

bioRxiv2015

A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5' m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5'-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell.\n\nFor murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of [~]20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.

Molecular Biology↗

bioRxiv2015

TET1 Modulates H4K16 Acetylation by Interacting with hMOF to Regulate Expression of DNA Repair Genes and Oncogenic Transformation

The Ten Eleven Translocation 1 (TET1) protein is a DNA demethylase that regulates gene expression through alteration of DNA methylation. Recent studies have demonstrated that TET1 could modulate transcriptional expression independent of its DNA demethylation activity; however, the detailed mechanisms underlying TET1s role in such transcriptional regulation remain not well understood. Here, we uncovered that Tet1 formed a chromatin complex with histone acetyltransferase Mof and scaffold protein Sin3a in mouse embryonic stem cells by integrative genomic analysis using publicly available ChIP-seq data sets. Specifically, the TET1/SIN3A/hMOF complex mediates acetylation of histone H4 at lysine 16, via facilitating the binding of hMOF on chromatin, to regulate expression of important DNA repair genes in DNA double strand breaks, including TP53BP1, RAD50, RAD51, and BRCA1, for homologous recombination and non-homologous end joining repairs. Under hydrogen peroxide-induced DNA damage, dissociation of TET1 and hMOF from chromatin, concurrent with increased binding of SIRT1 on chromatin, led to hypo-acetylation of H4K16, reduced expression of these DNA repair genes, and DNA repair defects in a DNA methylation independent manner. A similar epigenetic dynamic alteration was also observed in H-RASV12 oncogenic-transformed cells, supporting the notion that suppression of TET1 downregulates DNA repair genes through modifying H4K16ac, instead of its demethylation function, and therefore contribute to tumorigenesis. Taken together, our results suggested a mechanistic link between a novel TET1 complex and H4K16ac, DNA repair genes expression, and genomic instability.

Molecular Biology↗

bioRxiv2015

A novel recombinant DNA system for high efficiency affinity purification of proteins in Saccharomyces cerevisiae

AbstractIsolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. The tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this we have created a recombinant \"CelTag\" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background in a single step. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent will allow isolation of rare or unstable protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or chromatin-associated assemblies.

Molecular Biology↗

bioRxiv2015

Sampling the conformational space of the catalytic subunit of human γ-secretase

Human {gamma}-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimers disease. Our previous cryo-EM structure of {gamma}-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we introduce an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of {gamma}-secretase. In addition, we present a {gamma}-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

Molecular Biology↗

bioRxiv2015

Targeted reduction of highly abundant transcripts with pseudo-random primers

Transcriptome studies based on quantitative sequencing estimate gene expression levels by measuring the abundance of target RNAs in libraries of sequence reads. The sequencing cost is proportional to the total number of sequenced reads. Therefore, in order to cover rare RNAs, considerable quantities of abundant and identical reads have to be sequenced. This major limitation can be lifted by strategies used to deplete the library from some of the most abundant sequences. However, these strategies involve either an extra handling of the input RNA sample, or the use of a large number of reverse-transcription primers (termed \"not-so-random primers\"), which are costly to synthetize and customize. Here, we demonstrate that with a precise selection of only 40 \"pseudo-random\" reverse-transcription primers, it is possible to decrease the rate of undesirable abundant sequences within a library without affecting the transcriptome diversity. \"Pseudo-random\" primers are simple to design, and therefore are a flexible tool for enriching transcriptome libraries in rare transcripts sequences.

Molecular Biology↗

bioRxiv2015

Stochastic Analysis Of An Incoherent Feedforward Genetic Motif

Gene products (RNAs, proteins) often occur at low molecular counts inside individual cells, and hence are subject to considerable random fluctuations (noise) in copy number over time. Not surprisingly, cells encode diverse regulatory mechanisms to buffer noise. One such mechanism is the incoherent feedforward circuit. We analyze a simplistic version of this circuit, where an upstream regulator X affects both the production and degradation of a protein Y. Thus, any random increase in Xs copy numbers would increase both production and degradation, keeping Y levels unchanged. To study its stochastic dynamics, we formulate this network into a mathematical model using the Chemical Master Equation formulation. We prove that if the functional dependence of Ys production and degradation on X is similar, then the steady-distribution of Ys copy numbers is independent of X. To investigate how fluctuations in Y propagate downstream, a protein Z whose production rate only depend on Y is introduced. Intriguingly, results show that the extent of noise in Z increases with noise in X, in spite of the fact that the magnitude of noise in Y is invariant of X. Such counter intuitive results arise because X enhances the time-scale of fluctuations in Y, which amplifies fluctuations in downstream processes. In summary, while feedforward systems can buffer a protein from noise in its upstream regulators, noise can propagate downstream due to changes in the time-scale of fluctuations.

Molecular Biology↗

bioRxiv2015

Augmentation of nonsense mediated decay by rapamycin

RNA surveillance by the Nonsense Mediated Decay (NMD) pathway eliminates potentially deleterious transcripts containing Premature Termination Codons (PTCs). The transition from a pioneering round of translation to steady state translation is hypothesized to be a major checkpoint in this process. One hallmark of mRNAs licensed for translation is the exchange of 7-methylguanosine cap binding proteins. However, mRNAs undergoing steady state translation are also NMD substrates, raising mechanistic questions about the NMD checkpoint. To test the role of cap binding proteins in NMD, we modulated the protein composition of cytoplasmic messenger ribonucleoprotein particles (mRNPs) with the naturally occurring macrolide rapamycin. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD. Rapamycin-treatment significantly reduces the levels of endogenous and exogenous PTC-containing mRNA isoforms in a dose- and UPF1- dependent manner. PTC-containing transcripts exhibit a shorter half-life upon rapamacyin-treatment as compared to non-PTC isoforms. Rapamycin also causes depletion of PTC-containing mRNA isoforms from polyribosomes, suggesting that actively translating ribosomes can transition between low and high NMD states. Importantly, mRNPs show depletion of eIF4E and retention of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin potentiates pioneer-like mRNP context thereby decreasing NMD evasion.

Molecular Biology↗

bioRxiv2015

Sub-tomogram averaging in RELION

Electron cryo-tomography (cryo-ET) and sub-tomogram averaging allow structure determination of macromolecules in situ, and are gaining in popularity for initial model generation for single-particle analysis. We describe herein, a protocol for sub-tomogram averaging from cryo-ET data using the RELION software. We describe how to calculate newly developed three-dimensional models for the contrast transfer function and the missing wedge of each sub-tomogram, and how to use these models for regularized-likelihood refinement. This approach has been implemented in the existing workflow for single-particle analysis, so that users may conveniently tap into existing capabilities of the RELION software. As example applications, we present analyses of purified hepatitis B capsid particles and S. cerevisiae 80S ribosomes. In both cases, we show that following initial classification, sub-tomogram averaging in RELION allows de novo generation of initial models, and provides high-resolution maps where secondary structure elements are resolved.

Molecular Biology↗

bioRxiv2015

A phylogenetically diverse class of blind type 1 opsins

Opsins are photosensitive proteins catalyzing light-dependent processes across the tree of life. For both microbial (type 1) and metazoan (type 2) opsins, photosensing depends upon covalent interaction between a retinal chromophore and a conserved lysine residue. Despite recent discoveries of potential opsin homologs lacking this residue, phylogenetic dispersal and functional significance of these abnormal sequences have not yet been investigated. We report discovery of a large group of putatively non-retinal binding opsins, present in a number of fungal and microbial genomes and comprising nearly 30% of opsins in the Halobacteriacea, a model clade for opsin photobiology. Based on phylogenetic analyses, structural modeling, genomic context and biochemistry, we propose that these abnormal opsin homologs represent a novel family of sensory opsins which may be involved in taxis response to one or more non-light stimuli. This finding challenges current understanding of microbial opsins as a light-specific sensory family, and provides a potential analogy with the highly diverse signaling capabilities of the eukaryotic G-protein coupled receptors (GPCRs), of which metazoan type 2 opsins are a light-specific sub-clade.

Molecular Biology↗

bioRxiv2015

A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells

Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionary conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD - an abundant and highly conserved cytoplasmic lncRNA. Most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two Pumilio homologs in mammals. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA binding proteins, an activity which positions them at key junctions of cellular signaling pathways.

Molecular Biology↗

bioRxiv2016

Identification of physical interactions between genomic regions by enChIP-Seq

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. However, the methods for confidently detecting physical interactions between genomic regions remain limited. Here, we demonstrate the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9) and a single guide RNA (sgRNA). Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5HS5 locus, which regulates expression of the {beta}-globin genes, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism directly or indirectly mediated by physical interactions between these loci. Our data suggest that enChIP-Seq is a potentially useful tool for detecting physical interactions between genomic regions in a non-biased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

Molecular Biology↗

bioRxiv2016

Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites

Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence \"reads\" that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files.

Molecular Biology↗

bioRxiv2016

Development of molecular markers for determining continental origin of wood from white oaks (Quercus L. sect. Quercus)

To detect and avoid illegal logging of valuable tree species, identification methods for the origin of timber are necessary. We used next-generation sequencing to identify chloroplast genome regions that differentiate the origin of white oaks from the three continents; Asia, Europe, and North America. By using the chloroplast genome of Asian Q. mongolica as a reference, we identified 861 variant sites (672 single nucleotide polymorphisms (SNPs); 189 insertion/deletion (indel) polymorphism) from representative species of three continents (Q. mongolica from Asia; Q. petraea and Q. robur from Europe; Q. alba from North America), and we identified additional chloroplast polymorphisms in pools of 20 individuals each from Q. mongolica (789 variant sites) and Q. robur (346 variant sites). Genome sequences were screened for insertion/deletion (indel) polymorphisms to develop markers that identify continental origin of oak species, and that can be easily evaluated using a variety of detection methods. We identified five indel and one SNP that reliably identify continent-of-origin, based on evaluations of up to 1078 individuals representing 13 white oak species and three continents. Due to the size of length polymorphisms revealed, this marker set can be visualized using capillary electrophoresis or high resolution gel (acrylamide or agarose) electrophoresis. With these markers, we provide the wood trading market with an instrument to comply with the U.S. and European laws that require timber companies to avoid the trade of illegally harvested timber.

Molecular Biology↗

bioRxiv2016

Osteosarcoma with apparent Ewing sarcoma gene rearrangement

Poorly differentiated round cell sarcomas present diagnostic challenges due to their variable morphology and lack of specific immunophenotypic markers. We present a case of a 15-year-old female with a tibial tumor that exhibited features of Ewing-like sarcoma, including apparent rearrangement of the EWSR1 gene. Hybridization capture-based next-generation DNA sequencing showed evidence of complex genomic rearrangements, absence of known pathogenic Ewing-like chromosome translocations, and deletions RB1, PTCH1, and ATRX, supporting the diagnosis of osteosarcoma. This illustrates the potential of clinical genomic profiling to improve diagnosis and enable specifically targeted therapies for cancers with complex pathologies.

Molecular Biology↗

bioRxiv2016

Regulating telomere length from the inside out: The replication fork model

Telomere length is regulated around an equilibrium set point. Telomeres shorten during replication and are lengthened by telomerase. Disruption of the length equilibrium leads to disease, thus it is important to understand the mechanisms that regulate length at the molecular level. The prevailing protein counting model for regulating telomerase access to elongate the telomere does not explain accumulating evidence of a role of DNA replication in telomere length regulation. Here I present an alternative model: the replication fork model that can explain how passage of a replication fork and regulation of origin firing affect telomere length.

Molecular Biology↗

bioRxiv2016

Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome

Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed, for the first time, a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and provided evidence for a collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5LTR promoter.

Molecular Biology↗

bioRxiv2016

KlebSeq: A Diagnostic Tool for Healthcare Surveillance and Antimicrobial Resistance Monitoring of Klebsiella pneumoniae

Healthcare-acquired infections (HAIs) kill tens of thousands of people each year and add significantly to healthcare costs. Multidrug resistant and epidemic strains are a large proportion of HAI agents, and multidrug resistant strains of Klebsiella pneumoniae, a leading HAI agent, have become an urgent public health crisis. In the healthcare environment, patient colonization of K. pneumoniae precedes infection, and transmission via colonization leads to outbreaks. Periodic patient screening for K. pneumoniae colonization has cost-effective and life-saving potential. In this study, we describe the design and validation of KlebSeq, a highly informative screening tool that detects Klebsiella species and identifies clinically important strains and characteristics using highly multiplexed amplicon sequencing without a live culturing step. We demonstrate the utility of this tool on several complex specimen types including urine, wound swabs and tissue, several types of respiratory, and fecal, showing K. pneumoniae species and clonal group identification and antimicrobial resistance and virulence profiling, including capsule typing. Use of this amplicon sequencing tool can be used to screen patients for K. pneumoniae carriage to assess risk of infection and outbreak potential, and the expansion of this tool can be used for several other HAI agents or applications.

Molecular Biology↗