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Results for “bioinformatics”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2022

Did Caenorhabditis nematodes recycle transposons to fight pathogens?

Animal genomes consist largely of sequences derived from transposons which were previously considered as junk DNA and active transposons can even be deleterious for organismal fitness. Nevertheless, the huge pool of transposon-derived sequences also forms the raw material for molecular innovations. Here, we follow up on the incidental finding of a transposon-derived DNA binding domain in a subset of F-box genes in Caenorhabditis elegans. Based on phylogenetic analysis, we show that a single gene fusion followed by individual losses explains most members of this novel gene family. Phylogenomic data of available Caenorhabditis genomes allowed us to trace this fusion event to the ancestor of the Elegans group. Additional homology searches suggest endogenous Mariner transposons as the likely source of the coopted sequence. Further bioinformatic characterization of different F-box families by Gene Ontology analysis, gene module comparisons, and literature research identified first evidence that some F-box genes might be involved in innate immunity, as it had been proposed previously based on adaptive signatures of molecular evolution. Specifically, the F-box gene fbxa-158 contains one of the transposon-derived domains and was shown to interact with the components of the intracellular pathogen response machinery targeting Microsporidia and viruses. Thus, cooption of transposon-derived sequences likely contributed to the adaptive evolution of the F-box superfamily in Caenorhabditis nematodes. Significance statementWhen considering transposons as genomic junk or selfish elements that are detrimental for organismal fitness, we often neglect the potential of transposon-derived sequences as a source of molecular innovation. Here, we characterize a case where a transposon-derived sequence has been coopted by one of the largest and fastest evolving gene superfamilies in Caenorhabditis nematodes which include the model organism C. elegans. The resulting chimeric gene family has been stably maintained for about 20 million years and bioinformatic analysis reveals first evidence for potential functions in innate immunity. Thus, strong evolutionary pressure might have forced Caenorhabditis nematodes to recycle transposons in order to fight pathogens.

evolutionary biology↗

bioRxiv2022

NGS data reveals the phyllosphere microbiome of wheat plants infected by the fungal pathogen Zymoseptoria tritici

The fungal pathogen Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), a major wheat disease in Western Europe. Microorganisms inhabiting wheat leaves might act as beneficial, biocontrol or facilitating agents that could limit or stimulate the development of Z. tritici. Improving our understanding of microbial communities in the wheat phyllosphere would lead to new insights into STB management. This resource announcement provides fungal and bacterial metabarcoding datasets obtained by sampling wheat leaves differing in the presence of symptoms caused by Z. tritici. Tissues were sampled from three wheat commercial varieties on three sampling dates during a cropping season. Weeds around wheat fields were sampled as well. In total, more than 450 leaf samples were collected. The pathogen Z. tritici was quantified using qPCR. We provide the raw metabarcoding datasets, the Amplicon Sequence Variant (ASV) tables obtained after bioinformatic processing, the metadata associated to each sample (sampling date, wheat variety and tissue health condition), a preliminary descriptive analysis of the data, and the code used for bioinformatic and descriptive statistical analysis.

microbiology↗

bioRxiv2022

Single-cell profiling of lncRNAs in human germ cells and molecular analysis reveals transcriptional regulation of LNC1845 on LHX8

Non-coding RNAs exert diverse functions in many cell types. In addition to transcription factors from coding genes, non-coding RNAs may also play essential roles in shaping and directing the fate of germ cells. Here, we report the presence of many long non-coding RNAs (lncRNAs) which were specifically expressed in the germ cells during human gonadal development by single-cell profiling of the reported datasets, and the functional characterization of one divergent lncRNA, LNC1845. Comprehensive bioinformatic analysis of these lncRNAs indicates that divergent lncRNAs occupied the majority of female and male germ cells. Integrating lncRNA expression into the bioinformatic analysis also enhances the cell-type classification of female germ cells. Functional dissection using in vitro differentiation of human pluripotent stem cells to germ cells revealed the regulatory role of LNC1845 on a transcription factor essential for ovarian follicle development, LHX8, by modulating the levels of histone modifications, H3K4me3 and H3K27Ac. Hence, this study provides a comprehensive analysis of lncRNAs in developing germ cells and elucidates how a lncRNA function as a cis regulator during human germ cell development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486705v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@31290forg.highwire.dtl.DTLVardef@17ec505org.highwire.dtl.DTLVardef@1b8cd0dorg.highwire.dtl.DTLVardef@19e4de3_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIlncRNAs are expressed at significantly higher levels in both male and female germ cells than in somatic cells during human gonadal development. C_LIO_LIDivergent lncRNAs are enriched in the female meiotically and male mitotically arrested germ cells. C_LIO_LILNC1845 regulates the transcriptional expression of LHX8 by modulating H3K4me3 and H3K27Ac levels. C_LI

developmental biology↗

bioRxiv2022

Multi-Omics Regulatory Network Inference in the Presence of Missing Data

A key problem in systems biology is the discovery of regulatory mechanisms that drive phenotypic behaviour of complex biological systems in the form of multi-level networks. Modern multi-omics profiling techniques probe these fundamental regulatory networks but are often hampered by experimental restrictions leading to missing data or partially measured omics types for subsets of individuals due to cost restrictions. In such scenarios, in which missing data is present, classical computational approaches to infer regulatory networks are limited. In recent years, approaches have been proposed to infer sparse regression models in the presence of missing information. Nevertheless, these methods have not been adopted for regulatory network inference yet. In this study, we integrated regression-based methods that can handle missingness into KiMONo, a Knowledge guIded Multi-Omics Network inference approach, and benchmarked their performance on commonly encountered missing data scenarios in single- and multi-omics studies. Overall, two-step approaches that explicitly handle missingness performed best for a wide range of random- and block-missingness scenarios on imbalanced omics-layers dimensions, while methods implicitly handling missingness performed best on balanced omics-layers dimensions. Our results show that robust multi-omics network inference in the presence of missing data with KiMONo is feasible and thus allows users to leverage available multi-omics data to its full extent. Juan Henao is a 3rd year PhD candidate at Computational Health Center at Helmholtz Center Munich working on multi-omics and clinical data integration using both, bulk and single-cell data. Michael Lauber is a PhD Candidate at the Chair of Experimental Bioinformatics at the Technical University Munich. Currently, he is working on an approach for inference of reprogramming transcription factors for trans-differentiation. Manuel Azevedo is a Masters student at the Technical University of Munich in Mathematics with a focus on Biomathematics and Biostatistics. Currently, he is working as a Student Assistant at Helmholtz Munich, where he is also doing his masters thesis. Anastasiia Grekova is a Masters student of bioinformatics at the Technical University of Munich and the Ludwig-Maximilians-University Munich, working on multi-omics data integration in Marsico Lab at HMGU. Fabian Theis is the Head of the Institute of Computational Biology and leading the group for Machine Learning at Helmholtz Center Munich. He also holds the chair of Mathematical modelling of biological systems, Department of Mathematics, Technical University of Munich as an Associate Professor. Markus List obtained his PhD at the University of Southern Denmark and worked as a postdoctoral fellow at the Max Planck Institute for Informatics before starting his group Big Data in BioMedicine at the Technical University of Munich. Christoph Ogris holds a PostDoc position in the Marsico Lab at Helmholtz-Center Munich. His research focuses on predicting and exploiting multi-modal biological networks to identify disease-specific cross-omic interactions. Benjamin Schubert obtained his PhD at the University of Tubingen and worked as a postdoctoral fellow at Harvard Medical School and Dana-Farber Cancer Institute USA before starting his group for Translational Immmunomics at the Helmholtz Center Munich.

systems biology↗

bioRxiv2022

Circulating U13 small nucleolar RNA as a candidate biomarker for Huntington's disease

Background and ObjectivesFluid biomarkers are a recent field of interest in Huntington disease (HD). We focused on small circulating RNAs from plasma of subjects with prodromal (pre-HD) and overt disease by a two-stage approach: an unbiased investigation by an array method and a validation study to quantify a significant small nucleolar RNA. MethodsThrough Affymetrix Gene-Chip-miRNA-Array we performed an exploratory study on 9 HD patients, 8 healthy subjects (HS) and 5 psychiatric patients (PP; who share drugs with HD patients, to control for iatrogenic effects). Through real time PCR we validated the results in an independent population of 24 HD patients, 15 pre-HD, 24 PP, 28 Alzheimers disease (AD) patients (added to control the disease-specificity of our finding) and 23 HS. A bioinformatic analysis was also performed to interpret our finding. ResultsThe microarray results showed a significant signal for U13 small nucleolar RNA (SNORD13) that was increased in plasma of HD patients compared to controls (fold change, 1.54, p =0.003 HD vs. HS, and fold change 1.44 p = 0.0026 HD vs. PP). In the validation population the significant increase in HD patients was evident compared to both pre-HD and the three control groups (p<0.00001). The plasma levels of SNORD13 correlated with the status of mutant huntingtin carrier and the disease duration (respectively R=0.69; p<0.000001; R=0.49; p=0.015). Through receiver operating characteristic (ROC) curve analysis, we showed high accuracy of plasmatic SNORD13 in discriminating HD patients from pre-HD and control groups (AUC=0.963), outperforming values reported in another study for intrathecal or plasmatic mutant huntingtin and neurofilament light chain as biomarkers of overt HD. The bioinformatic analysis on SNORD13 interactome and pathway analysis showed enrichments for factors involved in nuclear functions beyond the ribosome biogenesis. DiscussionWe report the unprecedented finding of a potential role of small nucleolar RNAs in HD. Circulating SNORD13 seems a good biomarker for clinical purposes. It seems to be specific for HD and to peripherally report a plausible tipping point in the pathogenic cascade at neuronal level, possibly paving the way for new therapeutic targets.

neuroscience↗

bioRxiv2022

Widespread allele-specific topological domains in the human genome are not confined to imprinted gene clusters.

BackgroundThere is widespread interest in the three-dimensional chromatin conformation of the genome and its impact on gene expression. However, these studies frequently do not consider parent-of-origin differences, such as genomic imprinting, which result in monoallelic expression. In addition, genome-wide allele-specific chromatin conformation associations have not been extensively explored. There are few accessible bioinformatic workflows for investigating allelic conformation differences and these require pre-phased haplotypes which are not widely available. ResultsWe assembled a bioinformatic pipeline, "HiCFlow", which performs haplotype assembly and visualisation of parental chromatin architecture. We benchmarked the pipeline using prototype haplotype phased Hi-C data from GM12878 cells at three disease associated imprinted gene clusters. Using RC-HiC (Region Capture Hi-C) and Hi-C data from further human cell lines (1-7HB2, IMR-90, and H1-hESCs) we were able to robustly identify the known stable allele-specific interactions at the H19/IGF2 locus. Other imprinted loci (DLK1 and SNRPN) were more variable and there was no "canonical imprinted 3D structure", but we could detect allele-specific differences in A/B compartmentalisation. Genome-wide, when TADs were unbiasedly ranked according to their allele-specific contact frequencies, a set of "allele-specific TADs" (ASTADs) could be defined. These occurred in genomic regions of high sequence variation. In addition to imprinted genes, ASTADs were also enriched for allele-specific expressed (ASE) genes. We found loci in ASTADs that have not previously been identified as ASE such as the bitter taste receptors (TAS2Rs). ConclusionsThis study highlights the widespread differences in chromatin conformation between heterozygous loci and provides a new framework for understanding ASE.

genomics↗

bioRxiv2022

Development of an antibody panel for imaging mass cytometry to investigate cancer-associated fibroblast heterogeneity and spatial distribution in archival tissues

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.

cancer biology↗

bioRxiv2022

The Solvation of the E. coli CheY Phosphorylation SiteMapped by XFMS

The Escherichia coli CheY protein belongs to a large bacterial response regulator superfamily. X-ray hydroxy radical foot-printing with mass spectroscopy (XFMS) has shown that allosteric activation of CheY by its motor target triggers a concerted internalization of aromatic sidechains. We reanalyzed the XFMS data to compare polar versus non-polar CheY residue positions. The polar residues around and including the 57D phosphorylated site had an elevated hydroxy radical reactivity. Bioinformatic measures revealed that a water-mediated hydrogen bond network connected this ring of residues with the central 57D. These residues solvated 57D to energetically stabilize the apo-CheY fold. The abundance of these reactive residues was reduced upon activation. This result was supported by the bioinformatics and consistent with the previously reported activation-induced increase in core hydrophobicity. It further illustrated XFMS detection of structural waters. Direct contacts between the ring residues and the phosphorylation site would stabilize the aspartyl phosphate. In addition, we report that the ring residue, 18R, is a constant central node in the 57D solvation network and that 18R non-polar substitutions determine CheY diversity as assessed by its evolutionary trace in bacteria with well-studied chemotaxis. These results showcase the importance of structured water dynamics for phosphorylation-mediated signal transduction.

biochemistry↗

bioRxiv2022

Conserved intramolecular networks in GDAP1 are closely connected to CMT-linked mutations and protein stability

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices 3, 7, and 8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. Many CMT mutation sites are highlighted in the bioinformatics analyses focusing on sequence conservation and entropy, and the analyses support the outlier nature of GDAP1 in the GST superfamily. A central role for the 6-7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration.

biochemistry↗

bioRxiv2022

Comparative analysis of CRISPR off-target activity discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells

While CRISPR-based editing most often occurs at DNA sequences with perfect homology to the guide RNA (gRNA), unintended editing can occur at highly homologous regions (i.e., off-target (OT) sites). Due to the pace at which genome editing therapies are approaching clinical applications, there is an emerging need to define effective workflows for investigating OT editing effects. A number of homology-dependent, in silico-based prediction methods and wet lab-based empirical methods exist to investigate OT editing, but few have been subjected to analytical assessment or head-to-head comparison in human primary cells using an ex vivo editing process optimized for high-fidelity gene editing. Therefore, we sought to compare publicly available in silico tools (COSMID, CCTop, and Cas-OFFinder) as well as empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) in the context of ex vivo hematopoietic stem and progenitor cell (HSPC) editing. To do so, we edited CD34+ HSPCs using 11 different guide RNAs (gRNAs) complexed with HiFi Cas9, then performed targeted next-generation sequencing of ~200-site panels containing a range of nominated OT sites identified by in silico and empirical methods. We identified an average of 0.45 OT sites per gRNA at an indel detection limit of 0.5%. This study confirmed the marked improvement in specificity with HiFi Cas9 compared to wild-type Cas9 without compromising on-target activity when delivered as an RNP. Additionally, all HiFi Cas9 OT sites using a standard 20nt gRNA were identified by all OT detection methods with one exception (SITE-seq did not identify an OT generated by an AAVS1 gRNA). This resulted in high sensitivity for the majority of OT nomination tools, however due to the large number of false positives called by most methods, in silico-based COSMID and empirical methods DISCOVER-Seq and GUIDE-Seq attained the highest positive predictive value. We did not find the empirical methods identified off-target sites that were not also identified by bioinformatic methods when delivered as an RNP complex. Finally, this study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity as well as positive predictive value which would enable more efficient identification of potential off-target sites without compromising a thorough examination for any given gRNA.

genomics↗

bioRxiv2022

Deletion of Tac1 gene impact kinase phosphorylation involved in signaling pathways associated with pain

Pain in elderly persons is often not adequately treated, and current treatments may lead to poor outcomes. Therefore, new treatment strategies need to be developed based on a better understanding of the mechanisms underlying the development of chronic pain. Recent studies have shown that Tac1-/- mice display a significant decrease in nociceptive pain responses to moderate or intense stimuli but present no phenotypic changes following light or nonpainful stimuli. Moreover, the deletion of the Tac1 gene led to a deficit of opioid peptides, which are essential to endogenous pain control mechanisms. Thus, we investigated whether Tac1-/- mice show defective pain modulatory pathways by specifically profiling protein kinases in mice spinal cord using phosphoproteomics and bioinformatics. Protein phosphorylation is a key feature of the cellular regulatory mechanism, and phosphorylation status is related to the regulation and modulation of protein-protein binding. Bioinformatics analysis revealed that MAPK, tyrosine kinase, senescence, interleukin signaling, and TCR signaling are modulated in Tac1-/- mice. Interestingly, these processes are intimately linked with inflammatory responses leading to the release of cytokines and chemokines implicated in the interactions and communications between cells. They are key players involved in the initiation and persistence of pathologic pain. The absence of the Tac1 gene products may trigger a much wider cell response to compensate for the lack of important components of the nociceptive pain transmission system.

neuroscience↗

bioRxiv2022

A general approach to discover conformational epitope-directed binders for selective inhibition of protein proteolytic activation

Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type (WT) POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro and catalytic domains for four different matrix metalloproteases (MMPs), such as MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impairs cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2, that is known to transform it from a tumor suppressor to an oncoprotein. We believe the EDS method greatly facilitates the generation antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications. SignificanceWe have developed a highly efficient platform to facilitate the directed selection in vitro of antibodies to a wide range of functional epitopes on proteins. This method uses a bioinformatic program to guide mutations in the local site of interest to create a decoy antigen that can effectively remove antibodies not binding the site of interest by negative selection, followed by positive selection with the WT antigen to identify antibodies to the epitope of interest. We demonstrate the generality and versatility of this method by successfully producing functional antibodies to block specific proteolytically sensitive epitopes on five different proteins including enzymes important in cancer. The epitope-directed selection (EDS) approach greatly facilitates the identification of binders to specific sites of interest on proteins to probe function and as potential immunotherapeutics.

animal behavior and cognition↗

bioRxiv2022

Skeletal muscle DNA methylation and mRNA responses to a bout of higher versus lower load resistance exercise in previously trained men

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower-load (LL) versus higher-load (HL) resistance exercise. Trained college-aged males (n=11, 23{+/-}4 years old, 4{+/-}3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 hours, and 6 hours after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p<0.001), although total training volume (sets x reps x load) was not significantly different between bouts (p=0.571). Regardless of bout, more CpG site methylation changes were observed at 3-versus 6-hours post exercise (239,951 versus 12,419, respectively; p<0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3- and 6-hours following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3- versus 6-hours following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 hours following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 hours following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion", "MAPK signaling", and "PI3K-Akt signaling" were significantly affected at the 3- and 6-hour time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

molecular biology↗

bioRxiv2022

Metabolic Sensing of Extra-cytoplasmic Copper Availability via Translational Control by a Nascent Exported Protein

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extra-cellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that co-translational export of the protein CutF regulates translation of the down-stream cutO-encoded multi-copper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is co-translationally exported by the signal recognition particle pathway. Binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its co-translational export via its C-terminal ribosome stalling-like motif. This allows the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO, which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and often, are located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using co-translational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. ImportanceMetabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolism to changing environments by transcriptional regulation. Here, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a co-translationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows the unfolding of a downstream mRNA stem-loop and enables translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for integrating environmental signals into metabolic adaptation.

microbiology↗

bioRxiv2022

The RNA binding proteins hnRNP H and F regulate splicing of a MYC dependent HRAS exon in Prostate Cancer Cells

The Myc proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by Myc transcriptionally upregulates the core pre-mRNA splicing machinery and causes mis-regulation of alternative splicing. However, our understanding of how splicing changes are directed by Myc is limited. We performed a signaling pathway-guided splicing analysis to identify Myc dependent splicing events. These included an HRAS cassette exon repressed by Myc across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of hnRNP H gene expression with Myc hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, hnRNP F expression showed a positive correlation with Myc hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal new mechanisms for Myc-dependent regulation of splicing, and point to new possible therapeutic targets in prostate cancers. SIGNIFICANCE STATMENTMyc Transformation by the proto-oncogene c-Myc causes dysregulation of the pre-mRNA splicing reaction in cancer, but it is not known how mRNA isoform changes are directed by Myc. Here, we use bioinformatics to identify a splicing event in another proto-oncogene, HRAS, that is regulated by Myc across multiple tumor types. We identify new splicing regulators, hnRNPs H and F, that control this HRAS exon by binding to enhancer elements within its downstream intron. Additional pan-cancer bioinformatic analyses show hnRNP H expression to be anti- correlated with Myc hallmarks, consistent with the reduced splicing of the HRAS exon in Myc driven cancer. These findings uncover new mechanisms by which Myc can alter splicing in cancer cells and provide new molecular targets for potential therapeutics.

molecular biology↗

bioRxiv2022

The secretome of irradiated peripheral mononuclear cells attenuates hypertrophic skin scarring

BackgroundHypertrophic scars can cause pain, movement restrictions, and reduction of quality of life. Despite numerous options to tackle hypertrophic scarring, efficient therapies are still scarce, and cellular mechanisms are not well understood. Secreted factors from peripheral blood mononuclear cells (PBMCsec) were previously described for their beneficial effects in tissue regeneration. Here, we investigated the effects of PBMCsec on skin scarring in mouse models and human scar explant cultures at single cell resolution (scRNAseq). MethodsMouse wounds and scars were treated with PBMCsec either intradermally or topically. Human mature scars were treated with PBMCsec ex vivo in explant cultures. All experimental settings were analyzed by single cell RNA sequencing (scRNAseq). A variety of bioinformatics approaches were used to decipher gene regulation in the scRNAseq data sets. Components of the extracellular matrix (ECM) were investigated in situ by immunofluorescence. The effect of PBMCsec on myofibroblast differentiation and elastin expression was investigated by stimulating human primary fibroblasts with TGF{beta}. FindingsTopical and intradermal application of PBMCsec regulated the expression of a variety of genes involved in pro-fibrotic processes and tissue remodeling. Our bioinformatics approach identified elastin as a common linchpin of antifibrotic action in both, the mouse and human experimental setting. In vitro, we found that PBMCsec prevents TGF{beta}-mediated myofibroblast-differentiation and attenuates abundant elastin expression through non-canonical signaling inhibition. Furthermore, TGF{beta}-induced breakdown of elastic fibers was strongly inhibited by addition of PBMCsec. InterpretationTogether, we showed anti-fibrotic effect of PBMCsec on cutaneous scars in mouse and human experimental settings, suggesting PBMCsec as a novel therapeutic option to treat skin scarring. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSParacrine factors secreted from irradiated peripheral mononuclear cells (PBMCsec) show strong tissue regenerative properties in a variety of organs and are shown to enhance cutaneous wound healing. Whether PBMCsec shows anti-fibrotic properties on scar formation has not been investigated so far. Added value of this studyIn the present study, we were able to demonstrate that PBMCsec improves quality of developing and mature scars in mouse and human scar tissue. We found that PBMCsec is able to attenuate the expression of various genes, promoting scar formation and inhibit TGF{beta}-induced myofibroblast differentiation. Elastin and TXNIP were identified as a common linchpin of its anti-fibrotic action. Implications of all the available evidenceUsing in vivo, ex vivo, and in vitro models and analyses on a single-cell level, our study paves the way for clinical studies evaluating the use of PBMCsec for the treatment of human cutaneous scars.

cell biology↗

bioRxiv2022

Census of exposed aggregation-prone regions in proteomes

Loss of solubility usually leads to the detrimental elimination of protein function. In some cases, the protein aggregation is also required for beneficial functions. Given the duality of this phenomenon, it remains a fundamental question how natural selection controls the aggregation. The exponential growth of genomic sequence data and recent progress with in silico predictors of the aggregation allows approaching this problem by a large-scale bioinformatics analysis. Most of the aggregation-prone regions are hidden within the 3D structures and, therefore, they cannot realize their potential to aggregate. Thus, the most realistic census of the aggregation prone regions requires crossing aggregation prediction with information about the location of the natively unfolded regions. This allows us to detect so-called "Exposed Aggregation-prone Regions" (EARs). Here, we analyzed the occurrence and distribution of the EARs in 76 full reference proteomes from the three kingdoms of life. For this purpose, we used a bioinformatics pipeline, which provides a consensual result based on several predictors of aggregation. Our analysis revealed a number of new statistically significant correlations about the presence of EARs in different organisms, their dependence on protein length, cellular localizations, co-occurrence with short linear motifs, and the level of protein expression. We also obtained a list of proteins with the conserved aggregation-prone sequences for further experimental tests. Insights gained from this work led to a deeper understanding of the functional and evolutionary relations of the protein aggregation.

molecular biology↗

bioRxiv2023

Botrytis cinerea small RNAs are associated with tomato AGO1 and silence tomato target genes supporting cross-kingdom RNAi between the fungal pathogen B. cinerea and its tomato host

Cross-kingdom or cross-species RNA interference (RNAi) is broadly present in many interacting systems between microbes/parasites and their plant and animal hosts. A recent study by Qin et al. (2022) performed correlation analysis using global sRNA- and mRNA-deep sequencing data of cultured B. cinerea and B. cinerea-infected tomato leaves and claimed that cross-kingdom RNAi may not occur in B. cinerea-tomato interaction (Qin et al., 2022). Here, we use experimental evidence and additional bioinformatics analysis of the datasets produced by Qin et al. (2022) to identify the key reasons why a discrepancy between the conclusion of Qin et al. 2022 and previously published findings occurred. We also provided additional experimental evidence to support the presence of cross-kingdom RNAi between tomato and B. cinerea. We believe it is important to clarify the basic concept and mechanism of cross-kingdom/cross-species sRNA trafficking and illustrate proper bioinformatics analyses in this regard for all the scientists and researchers in this field.

plant biology↗