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bioRxiv2018

The RNA-binding protein AATF coordinates rRNA maturation

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

molecular biology↗

bioRxiv2018

Transcriptional responses of Biomphalaria pfeifferi and Schistosoma mansoni following exposure to niclosamide, with evidence for a synergistic effect on snails following exposure to both stressors

BackgroundSchistosomiasis is one of the worlds most common NTDs. Successful control operations often target snail vectors with the molluscicide niclosamide. Little is known about how niclosamide affects snails, including for Biomphalaria pfeifferi, the most important vector for Schistosoma mansoni in Africa. We used Illumina technology to explore how field-derived B. pfeifferi, either uninfected or harboring cercariae-producing S. mansoni sporocysts, respond to a sublethal exposure of niclosamide. This study afforded the opportunity to determine if snails respond differently to biotic or abiotic stressors, and if they reserve unique responses for when presented with both stressors in combination. We also examined how sporocysts respond when their snail host is exposed to niclosamide.\n\nPrincipal FindingsCercariae-producing sporocysts within snails exposed to niclosamide express ~68% of the genes in the S. mansoni genome, as compared to 66% expressed by intramolluscan stages of S. mansoni in snails not exposed to niclosamide. Niclosamide does not disable sporocysts nor does it seem to provoke from them distinctive responses associated with detoxifying a xenobiotic. For B. pfeifferi, niclosamide treatment alone increases expression of several features not up-regulated in infected snails including particular cytochrome p450s and heat shock proteins, glutathione-S-transferases, antimicrobial factors like LBP/BPI and protease inhibitors, and also provokes strong down regulation of proteases. Exposure of infected snails to niclosamide resulted in numerous up-regulated responses associated with apoptosis along with down-regulated ribosomal and defense functions, indicative of a distinctive, compromised state not achieved with either stimulus alone.\n\nConclusions/SignificanceThis study helps define the transcriptomic responses of an important and under-studied schistosome vector to S. mansoni sporocysts, to niclosamide, and to both in combination. It suggests the response of S. mansoni sporocysts to niclosamide is minimal and not reflective of a distinct repertoire of genes to handle xenobiotics while in the snail host. It also offers new insights for how niclosamide affects snails.\n\nAuthorS SummarySchistosomaisis control programs often employ the use of chemical molluscicides, such as niclosamide, to control the obligatory intermediate snail hosts. Despite its widespread use, we know little about how niclosamide affects snails like Biomphalaria pfeifferi, the most important vector Schistosoma mansoni in Africa. By sequencing the transcriptomes of uninfected and S. mansoni-infected B. pfeifferi exposed to niclosamide, we analyze the snails response to both biotic and abiotic stressors. We can also examine the response of S. mansoni to niclosamide exposure during intramolluscan development. Biomphalaria pfeifferi snails exposed only to niclosamide showed unique up-regulation of stress and defense-related transcripts not seen in snails infected with a biotic, like S. mansoni infection, alone. Schistosoma mansoni-infected B. pfeifferi exposed to niclosamide were clearly unable to regulate normal metabolic and detoxification processes. Cercariae-producing sporocysts within snails exposed to niclosamide are largely unaffected and continue to produce transcripts required for cercariae production.

molecular biology↗

bioRxiv2018

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of adults with poorly controlled asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 17A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFa, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, assessment of the circulating \"blood microbiome\" was performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in the plasma in a condition-specific manner, and many more were found to be expressed at altered levels. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthma state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

molecular biology↗

bioRxiv2018

Alternative interaction sites in the influenza A virus nucleoprotein mediate viral escape from the importin-α7 mediated nuclear import pathway

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Here, we addressed the question by which mechanisms influenza A viruses may escape restriction by the cellular importin-7 protein, a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Therefore, we assessed viral evolution in mice lacking the importin-7 gene. Here, we show that particularly three mutations occur with high frequency in the viral NP protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal and importin- isoforms. However, viral escape from importin-7 is, at least in part, associated with reduced replicative fitness in human cells.

molecular biology↗

bioRxiv2018

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

molecular biology↗

bioRxiv2018

Heat Shock in C. elegans Induces Downstream of Gene Transcription and Accumulation of Double-Stranded RNA

We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find antisense transcripts are generally increased after heat shock, and a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.

molecular biology↗

bioRxiv2018

Interplay between consensus and divergent RNA polymerase II C-terminal domain repeats in viability and targeting

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS, and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. To the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats fully supports Drosophila viability, but a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently-tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci independent of the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

molecular biology↗

bioRxiv2018

A piggyBac-based toolkit for inducible genome editing in mammalian cells

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

molecular biology↗

bioRxiv2018

Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding and molecular diagnostic techniques which are still under improvement even though the current SINE method for identification between An. coluzzii and An. gambiae works reliably. This study describes a refinement of the SINE method to increase sensitivity and high throughput method for the identification of both species and An. arabiensis using amplicon dissociation characteristics.\n\nField collected samples, laboratory reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were provided by the insectary of Vestergaard-NMIMR Vector Labs at the Noguchi Memorial Institute for Medical Research (Ghana) and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye.\n\nThe three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72 Celsius for An. arabiensis, 75{degrees}C for An. gambiae and 86{degrees}C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

molecular biology↗

bioRxiv2018

Unilateral relapse of Behcet’s disease-associated uveitis does not appear to cause asymmetric tear protein profiles

Purpose: To explore whether unilateral relapse of Bechets disease uveitis (BDU) causes differences in the tear proteome between the diseased and the contralateral quiescent eye.\n\nExperimental design: To minimize interindividual variations, bilateral tear samples were collected from the same patient (n=15) with unilateral relapse of BDU. A data-independent acquisition (DIA) strategy was used to identify proteins that differed between active and quiescent eyes.\n\nResults: A total of 1,797 confident proteins were identified in the tear samples, of which 371 are also highly expressed in various tissues and organs. Sixty-two (3.5%) proteins differed in terms of expression between tears in active and quiescent eyes, similar to the number of differentially expressed proteins (74, 4.1%) identified in a randomized grouping strategy. Furthermore, the intrapair trend of the differentially expressed proteins was not consistent and none of the proteins showed the same trend in more than 9 pairs of eyes.\n\nConclusions and clinical relevance: Unilateral relapse of BDU does not appear to cause asymmetric changes in the tear proteome between active and contralateral quiescent eyes. Tear fluid is a valuable source for biomarker studies of systemic diseases.\n\nStatement of clinical relevanceTears are an easily, noninvasively accessible body fluid that is a valuable source of biomarkers for various diseases. Behcets disease uveitis (BDU) has high potential to cause blindness and represents the leading cause of morbidity in BD patients, especially in frequently relapsing cases. Here, we adopted a method combining a \"dry\" method for tear preservation and nano-LC-DIA-MS/MS system to explore whether unilateral relapse of BDU causes differences in the tear proteome between the diseased and the contralateral quiescent eye, with the aim of evaluating tear fluid as a source for biomarker studies of uveitis relapse.

molecular biology↗

bioRxiv2018

Genetic population variation and phylogeny of Sinomenium acutum (Menispermaceae) in subtropical China through chloroplast marker

Sinomenium acutum (Menispermaceae) is a traditional Chinese medicine. In recent years, extensive harvesting for medicinal purposes has resulted in a sharp decline in its population. Genetic information is crucial for the proper exploitation and conservation of Sinomenium acutum, but little is known about it at present. In this study, we analyzed 77 samples from 4 populations using four non-coding regions (atpI-atpH, trnQ-5rps16, trnH-psbA, and trnL-trnF) of chloroplast DNA and 14 haplotypes (from C1 to C14) were identified. C1 and C3 were common haplotypes, which were shared by all populations, and C3 was an ancestral haplotype, the rest were rare haplotypes. Obvious phylogeographic structure was not existed inferred by GST / NST test. Mismatch distribution, Tajimas D and Fus FS tests failed to support a rapid demographic expansion in Sinomenium acutum. AMOVA highlighted that the high level of genetic differentiation within population. Low genetic variation among populations illustrated gene flow was not restricted. Genetic diversity analyses demonstrated that the populations of Xuefeng, Dalou, and Daba Mountains were possible refugia localities of Sinomenium acutum. Based on this study, we proposed a preliminary protection strategy for it that C1, C3, C11 and C12 must be collected. These results offer an valuable and useful information for this species of population genetic study as well as further conservation.

molecular biology↗

bioRxiv2018

Apicomplexan motility depends on the operation of an endocytic-secretory cycle

Apicomplexan parasites invade host cells in an active process, involving their ability to move by gliding motility and invasion. While the acto-myosin-system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions, such as how the force powering motility is generated. In many eukaryotes a secretory-endocytic cycle leads to recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility and generation of retrograde membrane flow. Here we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, since inhibition of endocytosis blocks retrograde flow and motility. We identified lysophosphatidic acid (LPA) as a potent stimulator of endocytosis and demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as nanogold particles. Furthermore, we show that surface proteins of the parasite are recycled during this process. Interestingly, the endocytic and secretory pathways of the parasite converge, and endocytosed material is subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings and we propose a novel model that reconciles parasite motility with observations in other eukaryotes: the fountain-flow-model for apicomplexan parasite motility.

molecular biology↗

bioRxiv2018

CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-{kappa}B signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-{kappa}B signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-{kappa}B signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-{kappa}B signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-{kappa}B signaling are discussed.

molecular biology↗

bioRxiv2018

Silencing cathepsin L expression reduces Myzus persicae protein content and the nutritional value as prey for Coccinella septempunctata

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control.\n\nHighlightsO_LISilencing expression of cathepsin L by RNA interference reduces protein content of Myzus persicae (green peach aphid) bodies.\nC_LIO_LIHoneydew of aphids with cathepsin L silenced contains elevated protein.\nC_LIO_LICathepsin L is required for efficient protein uptake from phloem sap.\nC_LIO_LIAphids with cathepsin L expression silenced have increased mortality and fewer offspring.\nC_LIO_LICoccinella septempunctata (seven-spotted ladybugs) grow more slowly on aphids with expression of cathepsin L silenced.\nC_LI\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC=\"FIGDIR/small/451211_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@5ee6d7org.highwire.dtl.DTLVardef@853fe8org.highwire.dtl.DTLVardef@26c2aborg.highwire.dtl.DTLVardef@b67e2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

Genome-wide gene expression profiling reveals that cuticle alterations and P450 detoxification are associated with pyrethroid resistance in Anopheles arabiensis populations from Ethiopia

BACKGROUNDVector control is the main intervention in malaria control and elimination strategies. However, the development of insecticide resistance is one of the major challenges for controlling malaria vectors. Anopheles arabiensis populations in Ethiopia showed resistance against both DDT and the pyrethroid deltamethrin. Although a L1014F target-site resistance mutation was present in the voltage gated sodium channel of investigated populations, the levels of resistance and biochemical studies indicated the presence of additional resistance mechanisms. In this study, we used genome-wide transcriptome profiling by RNAseq to assess differentially expressed genes between three deltamethrin and DDT resistant An. arabiensis field populations (Tolay, Asendabo, Chewaka) and two susceptible strains (Sekoru and Mozambique).\n\nRESULTSBoth RNAseq analysis and RT-qPCR showed that a glutathione-S-transferase, gstd3, and a cytochrome P450 monooxygenase, cyp6p4, were significantly overexpressed in the group of resistant populations compared to the susceptible strains, suggesting that the enzymes they encode play a key role in metabolic resistance against deltamethrin or DDT. Furthermore, a gene ontology enrichment analysis showed that expression changes of cuticle related genes were strongly associated with insecticide resistance, although this did not translate in increased thickness of the procuticle.\n\nCONCLUSIONOur transcriptome sequencing of deltamethrin/DDT resistant An. arabiensis populations from Ethiopia suggests non-target site resistance mechanisms and pave the way for further investigation of the role of cuticle composition in resistance.

molecular biology↗

bioRxiv2018

A small RNA controls bacterial resistance to gentamicin during iron starvation

Phenotypic resistance describes a bacterial population that becomes transiently resistant to an antibiotic without requiring a genetic change. We here investigated the role of the small regulatory RNA (sRNA) RyhB, a key contributor to iron homeostasis, in the phenotypic resistance of Escherichia coli to various classes of antibiotics. We found that RyhB induces resistance to gentamicin, an aminoglycoside that targets the ribosome, when iron is scarce. RyhB induced resistance is due to the inhibition of respiratory complexes Nuo and Sdh activities. These complexes, which contain numerous Fe-S clusters, are crucial for generating a proton motive force (pmf) that allows gentamicin uptake. RyhB directly represses the expression of nuo and sdh operons by binding to their mRNAs, thereby inhibiting their translation. Indirectly, RyhB also inhibits the maturation of Nuo and Sdh by repressing synthesis of the Isc Fe-S biogenesis machinery. Notably, our study identifies nuo as a new direct RyhB target and shows that respiratory complexes activity levels are predictive of the bacterial sensitivity to gentamicin. Altogether, these results unveil a new role for RyhB in the adaptation to antibiotic stress, an unprecedented consequences of its role in iron starvation stress response.\n\nAUTHORS SUMMARYUnderstanding the mechanisms at work behind bacterial antibiotic resistance has become a major health issue in the face of the antibiotics crisis. Here, we show that RyhB, a bacterial small regulatory RNA, induces resistance of Escherichia coli to the antibiotic gentamicin when iron is scarce, an environmental situation prevalent during host-pathogen interactions. This resistance is due to RyhB repression of the synthesis and post-translational maturation of the respiratory complexes Nuo and Sdh. These complexes are crucial in producing the proton motive force that allows uptake of the antibiotics in the cell. Altogether, these data point out to a major role for RyhB in escaping antibacterial action.

molecular biology↗

bioRxiv2018

Arginine methylation of DDX5 RGG/RG motif by PRMT5 regulates RNA/DNA resolution.

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often lead to catastrophic conflicts during replication resulting in DNA double strand breaks and genome instability. To prevent such conflicts, these hybrids require dissolution by helicases and/or RNaseH. Little information is known about how these helicases are regulated. Herein, we identify DDX5, an RGG/RG motif containing DEAD-box family of RNA helicase, as a crucial player in R-loop resolution. We define at the mechanistic level the function of DDX5 in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner leading to R-loop degradation by the XRN2 exoribonuclease. DDX5 deficient cells accumulated R-loops at loci known to form R-loops using RNA:DNA immunoprecipitation (DRIP)-qPCR and increased RNaseH sensitive RAD51 foci. PRMT5, an arginine methyltransferase, associated with DDX5 and methylated its RGG/RG motif. This motif was required to associate with XRN2 and resolve cellular R-loops. Furthermore, PRMT5 deficient cells accumulated R-loops, as detected by DRIP-qPCR resulting in increased gH2AX foci. Our findings define a new mechanism by which an RNA helicase, DDX5, is modulated by arginine methylation to resolve R-loops.

molecular biology↗

bioRxiv2018

Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by HslUV proteolysis

Whereas in eukaryotic cells, the Hsp90s are profusely-studied molecular chaperones controlling protein homeostasis together with Hsp70s, in bacteria, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild type E. coli, and from knockout mutants{Delta} dnaKdnaJ ({Delta}KJ),{Delta} htpG ({Delta}G) and{Delta} dnaKdnaJ{Delta}htpG ({Delta}KJG) and compared their growth rates under heat-stress also with{Delta} dnaKdnaJ{Delta}hslV. Whereas, expectedly, mutant {Delta}G showed no proteomic differences with wild-type, {Delta}KJ expressed more chaperones, proteases and ribosomes and dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that {Delta}KJG showed higher levels of metabolic and respiratory enzymes and both {Delta}KJG and{Delta} dnaKdnaJ{Delta}hslV grew better at 37oC than {Delta}KJ. The results indicate that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates, preferably by the HslUV protease.\n\nSignificance statementThe molecular chaperones Hsp70 and Hsp90 are among the most abundant and well-conserved proteins in all realms of life, forming together the core of the cellular proteostasis network. In eukaryotes, Hsp90 functions in collaboration with Hsp70; we studied this collaboration in E. coli, combining genetic studies with label-free quantitative proteomics in which both protein abundance and protein solubility were quantified. Bacteria lacking Hsp70 (DnaK) and its co-chaperone DnaJ ({Delta}dnaKdnaJ) grew slower and contained significantly less key metabolic and respiratory enzymes. Unexpectedly, an additional deletion of the Hsp90 (htpG) gene partially restored the WT phenotype. Deletion of the HslV protease in the {Delta}dnaKdnaJ background also improved growth, suggesting that bacterial Hsp90 mediates the degradation of Hsp70 substrates, preferentially through HslV.\n\nAt 37oC{Delta} dnaKdnaJ E. coli mutants grow slower than wild type cells. Quantitative proteomics shows that compared to wild type cells,{Delta} dnaKdnaJ cells grown at 30oC contain significantly less key metabolic and respiratory enzymes. Unexpectedly, deletion of the HtpG gene in the {Delta} dnaKdnaJ background ameliorates growth at 37oC and partially restores the cellular levels of some metabolic and respiratory enzymes.

molecular biology↗