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bioRxiv2018

An extracellular Argonaute protein mediates export of repeat-associated small RNAs into vesicles in parasitic nematodes

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.

molecular biology↗

bioRxiv2018

Development of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

O_LIThe crucian carp (Carassius carassius) is one of few fish species associated with small ponds in the UK. These populations contain genetic diversity not found in Europe and are important to conservation efforts for the species, which has declined across its range. Detection and monitoring of extant crucian carp populations are crucial for conservation success. Environmental DNA (eDNA) analysis could be very useful in this respect as a rapid, cost-efficient monitoring tool.\nC_LIO_LIWe developed a species-specific quantitative PCR (qPCR) assay for eDNA surveillance of crucian carp to enable non-invasive, large-scale distribution monitoring. We compared fyke netting and eDNA at ponds with (N = 10) and without (N = 10) crucian carp for presence-absence detection and relative abundance estimation, specifically whether DNA copy number reflected catch-per-unit-effort (CPUE) estimate. We examined biotic and abiotic influences on eDNA detection and quantification, and compared qPCR to standard PCR. Notably, eDNA occurrence and detection probabilities in relation to biotic and abiotic factors were estimated using a hierarchical occupancy model.\nC_LIO_LIeDNA analysis achieved 90% detection for crucian carp (N = 10), failing in only one pond where presence was known. We observed an overall positive trend between DNA copy number and CPUE estimate, but this was not significant. Macrophyte cover decreased the probability of eDNA occurrence at ponds, whereas CPUE and conductivity had positive and negative influences on eDNA detection probability in qPCR replicates respectively. Conductivity also had a negative effect on DNA copy number, but copy number increased with temperature and percentage of macrophyte cover. PCR was comparable to qPCR for species detection and may provide semi-quantitative information.\nC_LIO_LIOur results demonstrate that eDNA could enable detection of crucian carp populations in ponds and benefit ongoing conservation efforts, but imperfect species detection in relation to biotic and abiotic factors and eDNA workflow requires further investigation. Nonetheless, we have established an eDNA framework for crucian carp and sources of imperfect detection which future investigations can build upon.\nC_LI

molecular biology↗

bioRxiv2018

Analysis of seven putative Na+/H+ antiporters of Arthrospira platensis NIES-39 using transcription profiling and In-silico studies: an indication towards alkaline pH acclimation

Na+/H+ antiporters mediated pH regulation is one of the known mechanism(s), which advocates a possible role of the antiporters in the alkaline pH tolerance of Arthrospira platensis NIES-39 too. Seven putative Na+/H+ antiporters have been reported in A. platensis. We have characterized these seven antiporters, where A1, Q2, L2 and L6 belong to the CPA1 family whereas C5, D5 and O6 belong to CPA2 family through various In-silico analysis. Conserved domain analysis of these seven putative antiporters indicate the presence of nine different kinds of domains. Out of these nine domains, six domains function as monovalent cation-proton antiporters and two as the universal stress protein (Usp) category. Transcription profile of these seven antiporters was also generated at three different pH and time frames which showed a significant difference in the m-RNA levels at pH 7, 9 and at 11 along with a temporal pattern of the expression profile. The Insilico and the real time PCR analysis, put together, suggest the active participation of these seven putative Na+/H+ antiporters in alkaline pH homeostasis of this cyanobacterial strain where CPA1 subfamily antiporters play a major role.

molecular biology↗

bioRxiv2018

Transiently depleting RNPS1 leads to perdurable changes in alternative splicing

While robust regulatory mechanisms are expected to control the production of splice variants that confer distinct functions, a low level of stochasticity may be tolerated. To investigate stringency of regulation, we followed changes in the splicing of 192 alternative cassette exons after growth of cancer-derived HCT116 cells and embryonic colonocytes. In both cell lines approximately 15% of alternative splicing events changed by more than 10 percentage points over a 42-day period. We then carried out a cycle of transient depletions targeting RNPS1, a splicing regulator implicated in genomic stability. For alternative splicing units not regulated by RNPS1, the level of splicing changes was similar to the stochastic value obtained after normal growth. However, the frequency of perdurable changes was at least twice that value for splicing events regulated by RNPS1. A swap allele assay performed on four RNPS1-responsive units that underwent splicing changes indicated the presence of mutations mediating this effect. Specifically, a T to C mutation in a RNPS1-responsive exon of ADARB1 confered exon skipping. Our results suggest that fluctuations in the level of a splicing regulator preferentially impact the integrity of genes encoding transcripts that are regulated by this splicing factor to produce perdurable changes in alternative splicing. We discuss the potential implication of this process in human evolution.

molecular biology↗

bioRxiv2018

General decapping activators target different subsets of inefficiently translated mRNAs

The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and suggest that these factors may monitor mRNA translation and target unique features of individual mRNAs.

molecular biology↗

bioRxiv2018

An efficient and improved laboratory workflow and tetrapod database for larger scale eDNA studies

BackgroundThe use of environmental DNA, eDNA, for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the two most important risks of eDNA: sample contamination and taxonomic mis-assignment. These risks arise from the need for PCR amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FindingsOur high-throughput workflow minimises these risks via a four-step strategy: (1) technical replication with two PCR replicates and two extraction replicates; (2) using multi-markers (12S, 16S, CytB); (3) a twin-tagging, two-step PCR protocol;(4) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. As annotation errors in the reference sequences can result in taxonomic mis-assignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in over 50% of sequences being deleted from public reference databases, due to (1) limited overlap between our target amplicon and reference sequences; (2) mislabelling of reference sequences; (3) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate derived DNA (iDNA) dataset from 1532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples, but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criteria we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. ConclusionsOur metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider usage of e/iDNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

molecular biology↗

bioRxiv2018

Saracatinib and Dasatinib Fail To Prevent Heritable Pulmonary Arterial Hypertension

Evidence suggests that the deregulation of SRC Family Kinases may play a role in the development of heritable pulmonary arterial hypertension, associated with BMPR2 mutations. The truncated c-terminus of the BMPR2 protein is known to increase the phosphorylation and downstream activity of SRC tyrosine kinases. To test the hypothesis that the inhibition of SRC can prevent heritable PAH due to a BMPR2 mutation, we exposed BMPR2 mutant mice to SRC inhibitors, saracatinib and dasatinib, to block the SRC activation caused by the BMPR2 mutation. Saracatinib and dasatinib failed to prevent the development of PAH in BMPR2 mutant mice. Increased pressure in the right ventricle was not normalized and muscularization of large blood vessels was not reduced when compared to wild type mice. Inhibiting SRCs phosphorylation does not prevent heritable PAH, and thus supports evidence that SRCs aberrant localization and trafficking in PAH plays a more critical role in disease development.

molecular biology↗

bioRxiv2018

Stress-responsive and metabolic gene regulation are altered in low S-adenosylmethionine

S-adenosylmethionine (SAM) is the methyl donor that modifies proteins such as histones, nucleic acids and produces phosphatidylcholine. Thus variations in SAM levels could affect processes from lipogenesis to epigenetic gene regulation. SAM is hypothesized to link metabolism and chromatin modification, however, its role in acute gene regulation is poorly understood. We recently found that Caenorhabditis elegans with reduced SAM had deficiencies in bacterial-induced H3K4 trimethylation at selected pathogen-response genes, decreasing their expression and limiting survival on the pathogen Pseudomonas aeruginosa. This led us to the hypothesis that SAM may be generally required stress-responsive transcription. Here we show that C. elegans with low SAM fail to activate genome-wide transcriptional programs when exposed to bacterial or xenotoxic stress. However, heat shock responses were unaffected. We also investigated the role of two H3K4 methyltransferases that use SAM, set-2/SET1, and set-16/MLL and found that set-2/SET1 has a specific requirement in bacterial stress responses, whereas set-16/MLL was required for survival in all three stresses. These results define a role for SAM and H3K4 methyltransferases in the acute genome-wide remodeling of gene expression in response to stress. Finally, we find that the ability to modify metabolic gene expression correlates with enhanced survival in stress conditions.

molecular biology↗

bioRxiv2018

Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUURs SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

molecular biology↗

bioRxiv2018

Resilience of Atlantic Slippersnail Crepidula fornicata Larvae in the Face of Severe Coastal Acidification

Globally, average oceanic pH is dropping, and it will continue to decline into the foreseeable future. This ocean acidification (OA) will exacerbate the natural fluctuations in pH that nearshore ecosystems currently experience daily, potentially pushing marine organisms to their physiological limits. Adults of Crepidula fornicata (the Atlantic slippersnail) have proven remarkably resilient to many environmental changes, which is perhaps not surprising considering that they are common intertidally, have a geographically large native range, and have been extremely successful at invading coastal water in many other parts of the world. However, the larvae of C. fornicata have been shown to be somewhat more vulnerable than adults to the effects of reduced pH. Research to date has focused on the physiological impacts of OA on C. fornicata larvae; few studies have explored shifts in gene expression resulting from changes in pH. In the present study, we examined the response of young (4- day old) C. fornicata larvae to two extreme OA treatments (pH 7.5 and 7.6) relative to pH 8.0, documenting both phenotypic and genome-wide gene expression responses. We found that rearing larvae at reduced pH had subtle influences on gene expression, predominantly involving downregulation of genes related to growth and metabolism, accompanied by significantly reduced shell growth rates only for larvae reared at pH 7.5. Additionally, 10-day old larvae that had been reared at the two lower pH levels were far less likely to metamorphose within six hours when exposed to inducer. However, all larvae eventually reached similarly high levels of metamorphosis 24 hours after settlement induction. Finally, there were no observed impacts of OA on larval mortality. Taken together, our results indicate that far future OA levels have observable, but not severe, impacts on C. fornicata larvae, which is consistent with the resilience of this invasive snail across rapidly changing nearshore ecosystems. We propose that future work should delve further into the physiological and transcriptomic responses of all life history stages to gain a more comprehensive understanding of how OA impacts the intertidal gastropod C. fornicata.

molecular biology↗

bioRxiv2018

Rapid and sensitive virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. A highly sensitive amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described using both egg-propagated virus and clinical samples. 1D MinION sequencing of barcoded NDV amplicons was performed on 33 egg-grown isolates, (23 unique lineages, including 15 different NDV genotypes), and from 15 clinical swab samples from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. For all egg-grown samples, NDV was detected and virulence and genotype were predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes, of which the MinION method detected both genotypes in four of those samples. Additionally, testing a dilution series of one NDV sample resulted in detection of NDV with a 50% egg infectious dose (EID50) as low as 101 EID50/ml. This was accomplished in as little as 7 minutes of sequencing time, with a 98.37% sequence identity compared to the expected consensus. The high sensitivity, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for identification of NDV of different genotypes circulating worldwide. This general method will likely be applicable to other infections agents.

molecular biology↗

bioRxiv2018

LADL: Light-activated dynamic looping for endogenous gene expression control

Mammalian genomes are folded into tens of thousands of long-range looping interactions1,2. The cause and effect relationship between looping and genome function is poorly understood, and the extent to which chromatin loops are dynamic on short time scales remains a fundamental unanswered question. Currently available strategies for loop engineering involve synthetic transcription factors tethered to dCas93,4 or zinc fingers5,6, which are constitutively expressed5,6 or induced on long time scales by the presence of a small molecule3. Here we report a new class of 3-D optoepigenetic tools for the directed rearrangement of 3-D chromatin looping on short time scales using blue light. We create synthetic architectural proteins by fusing the CIBN protein subunit from Arabidopsis thaliana with enzymatically dead Cas9 (dCas9). We target our light-activated dynamic looping system (LADL) to two genomic anchors with CRISPR guide RNAs and engineer their spatial co-localization via light-induced heterodimerization of the cryptochrome 2 (CRY2) protein with dCas9-CIBN. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Looping changes occur as early as four hours after light induction. Using single molecule RNA FISH, we observe a LADL-induced increase in the total nascent Zfp462 transcripts and the number of Zfp462 alleles expressing simultaneously per cell. Moreover, LADL also increased synchronous Sox2 expression after reinforcement of a known Sox2-SE looping interaction. LADL facilitates loop synchronization across a large population of cells without exogenous chemical cofactors and can enable future efforts to engineer reversible and oscillatory looping on short time scales.

molecular biology↗

bioRxiv2018

A histone H4K20 methylation-mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. The mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here, we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase. Upon mitotic exit, appropriate chromatin relaxation is safeguarded by SET8-dependent methylation of histone H4 on lysine 20. Thus, in the absence of either SET8 or the H4K20 residue, substantial genome-wide chromatin decompaction occurs which allows excessive loading of the Origin Recognition Complex (ORC) in the daughter cells. ORC overloading stimulates aberrant recruitment of the MCM2-7 complex that promotes single-stranded DNA formation and DNA damage. Restoring chromatin compaction restrains excess replication licensing and the loss of genome integrity. Our findings identify a cell cycle-specific mechanism whereby fine-tuned chromatin relaxation suppresses excessive detrimental replication licensing and maintains genome integrity at the cellular transition from mitosis to G1 phase.

molecular biology↗

bioRxiv2018

CFTR misfolds during native-centric simulations due to entropic penalties of native state formation

Cystic fibrosis (CF) is a common genetic disorder that affects approximately 70,000 people worldwide. It is caused by mutation-induced defects in synthesis, folding, processing, or function of the Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR), a chloride-selective ion channel required for the proper functioning of secretory epithelia in tissues such as the lung, pancreas, and skin. The most common cause of CF is the single-residue deletion of F508 (F508del), a mutation present in one or both alleles in 90% of patients that induces severe folding defects and results in greatly reduced expression of the protein. Despite its medical importance, high-resolution mechanistic information about CFTR folding is lacking. In this study, we used molecular dynamics simulation with a native-centric force field to examine the folding and assembly of both full-length CFTR and the isolated first nucleotide-binding domain (NBD1). We observed that the protein was capable of substantial misfolding on both the intradomain and interdomain scale due to entropically favorable kinetic traps that exist on CFTRs folding free energy surface. These results suggest that even wild type CFTR, in the absence of any disease-related mutations, has suboptimal folding efficiency. We speculate that such entropically-driven misfolding also occurs in disease-prone mutants such as F508del and contributes to the proteins poor in vivo activity.

molecular biology↗

bioRxiv2018

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2-4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

molecular biology↗

bioRxiv2018

The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

Ancestral histones were present in organisms with small genomes, no nucleus, and little evidence for epigenetic regulation, suggesting histones may have additional older functions. We report that the histone H3-H4 tetramer is an enzyme that catalyzes the reduction of Cu2+ to Cu1+ when assembled in vitro from recombinant histones. Mutations of residues in the putative active site at the interface of the apposing H3 proteins alter the enzymatic activity and cellular processes such as Sod1 function or mitochondrial respiration that depend on availability of reduced copper. These effects are not due to altered gene expression or copper abundance but are consistent with decreased levels of cuprous ions. We propose that the H3-H4 tetramer is an oxidoreductase that provides biousable copper for cellular and mitochondrial chemistry. As the emergence of eukaryotes coincided with the Great Oxidation Event and decreased biousability of metals, the histone enzymatic function may have facilitated eukaryogenesis.

molecular biology↗

bioRxiv2018

Xist lncRNA forms silencing granules that induce heterochromatin formation and repressive complexes recruitment by phase separation

Main text Main text Microscopy RNA Structure Protein-RNA interactions Granule propensity Material and methods in... References Long non-coding RNAs (lncRNAs) are RNA molecules longer than 200 bases that lack coding potential1,2. They represent a significant portion of the cell transcriptome3 and work as activators or repressors of gene transcription acting on different regulatory mechanisms4-6. Indeed, lncRNAs can act as macro-scaffolds for protein recruitment7-14 and behave as guides and sponges for titrating RNA and proteins, influencing transcription at regulatory regions or triggering transcriptional interfere ...

molecular biology↗

bioRxiv2018

TTL proteins scaffold brassinosteroid signaling components at the plasma membrane to optimize signal transduction in plant cells

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. Here, we report that plant-specific TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are positive regulators of BR signaling functioning as scaffold for BR signaling components in Arabidopsis. TTL3 forms a complex with all core components involved in BR signaling, including the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1), the transcription factor BRASSINAZOLE RESISTANT1 (BZR1) and the phosphatase BRI1-SUPPRESSOR1 (BSU1), but excluding the co-receptor BAK1. TTL3 is mainly localized in the cytoplasm, but BR treatment increases its localization at the plasma membrane, where it strengthens the association with BR signaling components. Consistent with a role in BR signaling, mutations in TTL3 and related TTL1 and TTL4 genes cause reduced BR responsiveness. We propose a mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex, leading to the de-phosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1.

molecular biology↗