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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 595 records · Page 33Linked to original sources
bioRxiv2017

A Rice Dual-localized Pentatricopeptide Repeat Protein is involved in Organellar RNA Editing with MORFs

Flowering plants engage in diverse RNA editing events in mitochondrion and chloroplast on post-transcriptional process. Although several PPRs and MORFs were identified as RNA editing factors, the underlying mechanism of PPRs and the cooperation among them are still obscure. Here, we identified a rice dual-localized PPR mutant Ospgl1. Loss-of-function of OsPGLl resulted in defect of chloroplast RNA editing at ndhD-878 and mitochondrial RNA editing at ccmFc-543, which can be restored via complementary validation. Despite the synonymous editing on ccmFc-543, loss of editing at ndhD-878 caused failure of conversion from serine to leucine, leading to the dysfunction of chloroplast and defective in photosynthetic complex, further studies demonstrated OsPGL1 directly bound to both two transcripts. The interaction between three MORFs (MORF2/8/9) and OsPGL1 were confirmed in vitro and in vivo, implied OsPGL1 functioned on RNA editing via an editosome. It also suggested MORFs assisted and contributed to the flexible PPR-RNA recognition model during RNA editing through the cooperation with PPRs. These results provide new insight into the relationship between RNA editing and plant development on chloroplast.\n\nHighlightWe firstly characterized a dual-localized PPR protein which is required for RNA editing in mitochondrion and chloroplast simultaneously. OsPGL1 binds to two distinguish target transcripts directly and cooperated with MORFs.

molecular biology↗
bioRxiv2017

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Long non-coding RNAs (ncRNAs) are involved in both positive and negative regulation of transcription. Long ncRNAs are often enriched in the nucleus and at chromatin but whether chromatin-release plays a functional role is unknown. Here, we used epigenetic marks, expression level and strength of chromatin interactions to group long ncRNAs and find that those engaged in strong chromatin interactions are less enriched at chromatin in MCF-7 cells, suggesting a functional involvement of chromatin-release of long ncRNAs in transcriptional regulation. To study this further, we identify the long ncRNA A-ROD, an activating regulator of the Wnt signaling inhibitor DKK1. We show that A-ROD enhances transcription elongation of DKK1 in an RNA-dependent manner and that A-ROD recruits EBP1 to the DKK1 promoter. Our data suggest that the activating function depends on the release of A-ROD from chromatin, and further identify a functional regulatory interaction mediated by A-ROD in the transcription activation of DKK1. We propose that the release of a subset of long ncRNAs is important for their function, adding a new mechanistic perspective to the subcellular localization of long ncRNAs.

molecular biology↗
bioRxiv2017

Determinants of Transcription Initiation Directionality in Metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general and passive feature of all eukaryotic cis regulatory elements. To address this, we define promoters and enhancers in C. elegans, D. melanogaster and H. sapiens using ATAC-Seq and investigate the determinants of their transcription initiation directionalities by analyzing genome-wide nascent, cap-selected, polymerase run-on assays. All three species initiate divergent transcription from separate core promoter sequences. Sequence asymmetry downstream of forward and reverse initiation sites, known to be important for termination and stability in H. sapiens, is unique in each species. Chromatin states of divergent promoters are not entirely conserved, but in all three species, the levels of histone modifications on the +1 nucleosome are independent from those on the -1 nucleosome, arguing for independent initiation events. This is supported by an integrative model of H3K4me3 levels and core promoter sequence that is highly predictive of promoter directionality and of two types of promoters: those with balanced initiation directionality and those with skewed directionality. Lastly, D. melanogaster enhancers display variation in chromatin architecture depending on enhancer location, and D. melanogaster promoter regions with dual enhancer/promoter potential are enriched for divergent transcription. Our results point to a high degree of variation in regulatory element transcription initiation directionality within and between metazoans, and to non-passive regulatory mechanisms of transcription initiation directionality in those species.

molecular biology↗
bioRxiv2017

Binding of the integrated stress response inhibitor, ISRIB, reveals a regulatory site in the nucleotide exchange factor, eIF2B

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the {beta} and {delta} regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congeners in vivo and ISRIB-binding in vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

molecular biology↗
bioRxiv2017

Peripatric speciation associated with genome expansion and female-biased sex ratios in the moss genus Ceratodon

PREMISE OF THE STUDYA period of allopatry is widely believed to be essential for the evolution of reproductive isolation. However, strict allopatry may be difficult to achieve in some cosmopolitan, spore-dispersed groups, like mosses. Here we examine the genetic and genome size diversity in Mediterranean populations of the moss Ceratodon purpureus s.l. to evaluate the role of allopatry and ploidy change in population divergence.\n\nMETHODSWe sampled populations of the genus Ceratodon from mountainous areas and lowlands of the Mediterranean region, and from western and central Europe. We performed phylogenetic and coalescent analyses on sequences from five nuclear introns and a chloroplast locus to reconstruct their evolutionary history. We also estimated the genome size using flow cytometry, employing propidium iodide, and determined their sex using a sex-linked PCR marker.\n\nKEY RESULTSTwo well differentiated clades were resolved, discriminating two homogeneous groups: the widespread C. purpureus and a local group mostly restricted to the mountains in southern Spain. The latter also possessed a genome size 25% larger than the widespread C. purpureus, and the samples of this group consist entirely of females. We also found hybrids, and some of them had a genome size equivalent to the sum of the C. purpureus and Spanish genome, suggesting that they arose by allopolyploidy.\n\nCONCLUSIONSThese data suggest that a new species of Ceratodon arose via peripatric speciation, potentially involving a genome size change and a strong female-biased sex ratio. The new species has hybridized in the past with C. purpureus.

molecular biology↗
bioRxiv2017

Transcription activation domains of the yeast factors Met4 and Ino2: tandem activation domains with properties similar to the yeast Gcn4 activator

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation and chromatin modifications. Here we examine the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While AD function from both factors depends on hydrophobic residues, Ino2 further requires key conserved acidic and polar residues for optimal function. Binding studies show that the ADs bind multiple Med15 activator binding domains (ABDs) with a similar order of micromolar affinity, and similar but distinct thermodynamic properties. Protein crosslinking shows that no unique complex is formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with the yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

molecular biology↗
bioRxiv2017

Spotiton: New Features and Applications

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ~3 [A] resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

molecular biology↗
bioRxiv2017

Long-read sequencing of nascent RNA in S. pombe reveals coupling among RNA processing events

Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur co-transcriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe. Most multi-intron transcripts were fully spliced, consistent with rapid co-transcriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in Prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded co-transcriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of co-transcriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3 end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.

molecular biology↗
bioRxiv2017

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome where the Cas9 nuclease then induces a double stranded break (DSB)1,2. The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB3,4. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded donor oligonucleotide (ssODN) to the Cas9/guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease5, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to an 8-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci, and is more pronounced at low RNP concentrations.

molecular biology↗
bioRxiv2017

Molecular map of GNAO1-related disease phenotypes and reactions to treatment

The GNAO1 gene codes for the most commonly expressed G protein in the central nervous system. Pathogenic GNAO1 variants result in early-onset neurological phenotypes, sometimes with distinct epilepsy or movement disorder, and sometimes with both mani-festations in the same patient. The existing extensive knowledge about G-protein coupled receptor (GPCR) signaling provides the input needed to describe quantitatively how mutations modify the GPCR signal. This in turn allows rational interpretation of distinct phenotypes arising from mutations in GNAO1. In this work we outline a model that enables understanding of clinical phenotypes at a molecular level. The mutations affecting the catalytic pocket of GNAO1, we show, result in the improper withdrawal of the signal, and give rise to epileptic phenotypes (EPs). The converse is not true - some pure EPs are caused by mutations with no obvious impact on catalysis. Mutations close to the interface with GNAO1s downstream effector block the signal propagation in that direction, and manifest as a movement disorder phenotype without epilepsy. Quantifying the reported reaction to therapy highlights the tendency of the latter group to be unresponsive to the therapies currently in use. We argue, however, that the majority of clinically described mutations can impact several aspects of GNAO1 function at once, resulting in the continuum of phenotypes observed in patients. The reasoning based on GNAO1 signaling model provides a precision medicine paradigm to aid clinicians in selecting effective categories of medication, and in addition, can suggest pragmatic targets for future therapies.

molecular biology↗
bioRxiv2017

Results of follow-up experiments to "Odorant cues linked to social immunity induce lateralized antennal stimulation in honey bees (Apis mellifera L.)"

In 2017, we published the paper \"Odorant cues linked to social immunity induce lateralized antennal stimulation in honey bees (Apis mellifera L.)\" in Scientific Reports. Since then, we have performed three follow-up experiments which have either negative or contradictory results. Previously, we used electrophysiology to show that hygienic bees displayed significantly higher sensitivity to {beta}-ocimene when stimulated via their left antennae compared to their right. We repeated this assay using worker honey bees from a single hygienic colony and found, to our surprise, that the right antennae elicited higher sensitivity. We also previously attempted to identify a molecular basis for lateralization by using mass spectrometry-based proteomics to compare left and right antennal proteomes. Of the 1,845 proteins, none were differentially expressed. Here, we repeated this experiment but employed orthogonal peptide fractionation to increase proteome coverage to 3,114 proteins; however, still none were differentially expressed. Finally, we attempted to manipulate gene expression of a key antennal odorant binding protein linked to hygienic behaviour (OBP18) using RNA interference via antenna microinjection. We were not able to achieve long-lasting OBP18 knock-down, but comparing the proteomes of untreated, mock dsRNA-treated and OBP18 dsRNA-treated worker antennae revealed numerous off-target effects of the act of injecting alone. By openly reporting this data, we hope to set an example for information transparency.

molecular biology↗
bioRxiv2017

Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis

The success of Mycobacterium tuberculosis as a pathogen relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition of M. tuberculosis to dormancy and for emergence from the non-replicating persistent state. But these enzymes are double-edged swords, as their ability to degrade the cell wall, is potentially lethal to the bacterium itself. Hence, Rpf expression is tightly regulated. We have identified a novel regulatory element in the 5 untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which is restricted to pathogenic mycobacteria, suggesting a role in virulence. Moreover, we have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an RpfB antisense RNA. Finally, we show that rpfB is co-transcribed with downstream genes, ksgA and ispE. ksgA encodes a universally conserved methyl transferase involved in ribosome maturation and ispE encodes an essential ATP-dependent kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch. We propose that deregulation of this switch, associated with cell wall synthesis and ribosome function, presents a new target for anti-tuberculosis drug development.\n\nImportanceThis work describes the identification and characterisation of a novel regulatory RNA element/attenuator that controls cell wall synthesis and ribosome function in Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB). By switching between two different conformations, this RNA switch can either enable or inhibit transcription of a tri-cistronic mRNA that encodes a cell-wall remodelling enzyme crucial for activation of latent TB, an RNA methytransferase that is important for ribosome function and a protein kinase essential for early steps in cell wall synthesis. This RNA switch is only present in a subset of pathogenic mycobacteria, and by regulating the expression of three genes associated with classical antimicrobial targets we believe that it offers a novel important target for future anti-tuberculosis drugs.

molecular biology↗
bioRxiv2017

Isolation of nucleic acids from low biomass samples: detection and removal of sRNA contaminants

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. Due to its inherent instability, contamination with RNA is usually considered to be unlikely. Here we report the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and means for their depletion. Sequencing of sRNAs extracted from human plasma samples was performed and significant levels of non-human (exogenous) sequences were detected. The source of the most abundant of these sequences could be traced to the microRNA extraction columns by qPCR-based analysis of laboratory reagents. The presence of artefactual sequences originating from the confirmed contaminants were furthermore replicated in a range of published datasets. To avoid artefacts in future experiments, several protocols for the removal of the contaminants were elaborated, minimal amounts of starting material for artefact-free analyses were defined, and the reduction of contaminant levels for identification of bona fide sequences using ultra-clean extraction kits was confirmed. In conclusion, this is the first report of the presence of RNA molecules as contaminants in laboratory reagents. The described protocols should be applied in the future to avoid confounding sRNA studies.

molecular biology↗
bioRxiv2017

Characterisation and modelling of a polyhydroxyalkanoate synthase from Aquitalea sp. USM4 reveals a mechanism for polymer elongation

Polyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC from Aquitalea sp. USM4, PhaCAq, was characterised and showed a Km of 394 {micro}M and a kcat of 476.4 s-1 on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC from Cupriavidus necator, PhaCCn revealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model from Pseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180{degrees}. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAq model provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

molecular biology↗
bioRxiv2017

Adult-specific trimethylation of histone H3 lysine 4 is prone to dynamic changes with aging in C. elegans somatic cells

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Moreover, we found that global reduction of H3K4me3 levels results in significantly decreased RNA expression of genes that acquire H3K4me3 marking in their gene-bodies during adult stage, suggesting that altered H3K4me3 levels with age could result in age-dependent gene expression changes. Interestingly, the genes with dynamic changes in H3K4me3 and RNA levels with age are enriched for those involved in fatty acid metabolism, oxidation-reduction, and stress response. Therefore, our findings revealed divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on physiological relevance.

molecular biology↗
bioRxiv2017

Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here we present a simple method that combines the use of CRISPR-Cas9 and Cre-loxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next CRISPR-Cas9 is used to knockout an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate potential mutants of the tested gene.

molecular biology↗
bioRxiv2017

Spatial organization of single mRNPs at different stages of the gene expression pathway

mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while their sequestration into stress-granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localizing 5 and 3 ends, indicating that mRNAs are either not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs.\n\nOne Sentence SummarySingle mRNA studies in cells show RNA compaction changes depending on translational state, but mRNAs are not translated in closed-loop conformation.

molecular biology↗
bioRxiv2017

Deep sequencing reveals two Jurkat subpopulations with distinct miRNA profiles during camptothecin-induced apoptosis

microRNAs (miRNAs) are small non-coding RNAs of about 19-25 nt that regulate gene expression post-transcriptionally under various cellular conditions, including apoptosis. The miRNAs involved in modulation of apoptotic events in T cells are partially known. However, heterogeneity associated with cell lines makes it difficult to interpret gene expression signatures especially in cancer-related cell lines. Treatment of Jurkat T cell leukemia cell line with the universal apoptotic drug, camptothecin, resulted in identification of two Jurkat sub-populations: one that is sensitive to camptothecin and the other being rather intrinsically resistant. We sorted apoptotic Jurkat cells from the non-apoptotic ones prior to profiling miRNAs through deep sequencing. Our data showed that a total of 184 miRNAs were dysregulated. Interestingly, apoptotic and non-apoptotic sub-populations exhibited a distinct miRNA expression profile. In particular, 6 miRNAs were inversely expressed in these two sub-populations. The pyrosequencing results were validated by real time qPCR. Altogether these results suggest that miRNAs modulate apoptotic events in T cells and that cellular heterogeneity requires careful interpretation of miRNA expression profiles obtained from drug-treated cell lines.

molecular biology↗