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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Analysis of a structured intronic region of the LMP2 pre-mRNA from EBV reveals associations with human regulatory proteins.

Objective: The pre-mRNA of the Epstein-Barr virus LMP2 (latent membrane protein 2) has a region of unusual RNA structure that partially spans two consecutive exons and the entire intervening intron; suggesting RNA folding might affect splicing--particularly via interactions with human regulatory proteins. To better understand the roles of protein associations with this structured intronic region, we undertook a combined bioinformatics (motif searching) and experimental analysis (biotin pulldowns and RNA immunoprecipitations) of protein binding. Result: Characterization of the ribonucleoprotein composition of this region revealed several human proteins as interactors: regulatory proteins hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP U, HuR (human antigen R), and PSF (polypyrimidine tract-binding protein-associated splicing factor), as well as, unexpectedly, the cytoskeletal protein actin. Treatment of EBV-infected cells with drugs that alter actin polymerization specifically showed marked effects on splicing in this region. This suggests a potentially novel role for nuclear actin in regulation of viral RNA splicing.

molecular biology↗

bioRxiv2018

Relative Reduction of the Biological and phylogenetic diversity of oral microbiome in diabetic and pre-diabetic subjects

BackgroundThere is a suggested reciprocal relationship between oral health and systemic disease such as type 2 diabetes. In this relationship, a systemic disease predisposing to oral infection, and when that infection is present, the oral infection aggravates the progression of the systemic disease. Several studies suggested that some oral microbiome constituents are linked to both diabetes, metabolic syndrome and obesity. This study aims to compare the microbial diversity and population structure of oral microbiome among normoglycemic, impaired glucose tolerance (IGT) and diabetic subjects.\n\nMethodologyThis study followed a case-control design (15 T2D patients, 10 IGTs and, 19 controls). Patient records were screened as per the inclusion and exclusion criteria. Assessment of periodontitis and oral health was performed to all subjects. DNA Isolation purification and quantification from collected Saliva samples were performed. 16SrRNA hypervariable regions were amplified and sequenced. Generated sequences were subjected to bioinformatics analysis. Statistical analysis and diversity indices were computed with the statistical software R, the vegan R-package, and Past318 software.\n\nResultsA total observed number of 551 OTUs. A clear reduction of the number of species (OTUs) was observed in both IGT (412) and diabetic group (372) compared with the normoglycemic group (502). This was associated with a similar pattern of biological diversity among the three groups. Phylogenetic diversity (PD-SBL) value in the normoglycemic group was higher than the diabetic group. The diabetic group had the highest evenness value and the highest microbiome bacterial pathogenic content.\n\nConclusionWe observed a clear reduction in the biological and phylogenetic diversity in the diabetic and pre-diabetic oral microbiome in comparison with the normoglycemic oral microbiome. However, this reduction was associated with an increase in the pathogenic content of the hyperglycemic microbiomes.

microbiology↗

bioRxiv2018

Transcripts from multicopy gene families localizing to mouse Y long arm encode piRNAs and proteins

Heterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty, and Orly that are transcribed in testis. Of these Ssty and Sly genes encode proteins - yet all the copies of these RNAs are not translated. Using bioinformatic approaches, small RNA northern blots and electrophoretic mobility shift assays, we demonstrate here that these multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. Thus, we identified a piRNA cluster on mouse Y chromosome and also unraveled the dual role of Y-chromosome-encoded transcripts to act as primary transcripts of piRNAs in addition to their role as protein-coding RNAs.\n\nHIGHLIGHTSO_LIFirst report of a cluster of piRNAs on a mammalian Y chromosome\nC_LIO_LIReport of primary transcripts of piRNAs\nC_LIO_LIThese piRNAs putatively regulate autosomal genes expressed in mouse testis Ssty and Sly genes code for proteins as well as generate piRNAs\nC_LI

molecular biology↗

bioRxiv2018

Metagenomic profiling of ticks: identification of novel rickettsial genomes and detection of tick-borne canine parvovirus

BackgroundAcross the world, ticks act as vectors of human and animal pathogens. Ticks rely on bacterial endosymbionts, which often share close and complex evolutionary links with tick-borne pathogens. As the prevalence, diversity and virulence potential of tick-borne agents remain poorly understood, there is a pressing need for microbial surveillance of ticks as potential disease vectors.\n\nMethodology/Principal FindingsWe developed a two-stage protocol that includes 16S-amplicon screening of pooled samples of hard ticks collected from dogs, sheep and camels in Palestine, followed by shotgun metagenomics on individual ticks to detect and characterise tick-borne pathogens and endosymbionts. Two ticks isolated from sheep yielded an abundance of reads from the genus Rickettsia, which were assembled into draft genomes. One of the resulting genomes was highly similar to Rickettsia massiliae strain MTU5. Analysis of signature genes showed that the other represents the first genome sequence of the potential pathogen Candidatus Rickettsia barbariae. Ticks from a dog and a sheep yielded draft genome sequences of strains of the Coxiella-like endosymbiont Candidatus Coxeilla mudrowiae. A sheep tick yielded sequences from the sheep pathogen Anaplasma ovis, while Hyalomma ticks from camels yielded sequences belonging to Francisella-like endosymbionts. From the metagenome of a dog tick from Jericho, we generated a genome sequence of a canine parvovirus.\n\nSignificanceHere, we have shown how a cost-effective two-stage protocol can be used to detect and characterise tick-borne pathogens and endosymbionts. In recovering genome sequences from an unexpected pathogen (canine parvovirus) and a previously unsequenced pathogen (Candidatus Rickettsia barbariae), we demonstrate the open-ended nature of metagenomics. We also provide evidence that ticks can carry canine parvovirus, raising the possibility that ticks might contribute to the spread of this troublesome virus.\n\nAuthor SummaryWe have shown how DNA sequencing can be used to detect and characterise potentially pathogenic microorganisms carried by ticks. We surveyed hard ticks collected from domesticated animals across the West Bank territory of Palestine. All the ticks came from species that are also capable of feeding on humans. We detected several important pathogens, including two species of Rickettsia, the sheep pathogen Anaplasma ovis and canine parvovirus. These findings highlight the importance of hard ticks and the hazards they present for human and animal health in Palestine and the opportunities presented by high-throughput sequencing and bioinformatics analyses of DNA sequences in this setting.

microbiology↗

bioRxiv2018

A Type I Restriction-Modification System Associated with Enterococcus faecium Subspecies Separation

The gastrointestinal colonizer Enterococcus faecium is a leading cause of hospital-acquired infections. Multidrug-resistant (MDR) E. faecium are particularly concerning for infection treatment. Previous comparative genomic studies revealed that subspecies referred to as Clade A and Clade B exist within E. faecium. MDR E. faecium belong to Clade A, while Clade B consists of drug-susceptible fecal commensal E. faecium. Isolates from Clade A are further grouped into two sub-clades, A1 and A2. In general, Clade A1 isolates are hospital epidemic isolates whereas Clade A2 isolates are isolates from animals and sporadic human infections. Such phylogenetic separation indicates that reduced gene exchange occurs between the clades. We hypothesize that endogenous barriers to gene exchange exist between E. faecium clades. Restriction-modification (R-M) systems are such barriers in other microbes. We utilized bioinformatics analysis coupled with second generation and third generation deep sequencing platforms to characterize the methylome of two representative E. faecium strains, one from Clade A1 and one from Clade B. We identified a Type I R-M system that is Clade A1-specific, is active for DNA methylation, and significantly reduces transformability of Clade A1 E. faecium. Based on our results, we conclude that R-M systems act as barriers to horizontal gene exchange in E. faecium and propose that R-M systems contribute to E. faecium subspecies separation.\n\nIMPORTANCEEnterococcus faecium is a leading cause of hospital-acquired infections around the world. Rising antibiotic resistance in certain E. faecium lineages leaves fewer treatment options. The overarching aim of the attached work was to determine whether restriction-modification (R-M) systems contribute to the structure of the E. faecium species, wherein hospital-epidemic and non-hospital-epidemic isolates have distinct evolutionary histories and highly resolved clade structures. R-M provides bacteria with a type of innate immunity to horizontal gene transfer (HGT). We identified a Type I R-M system that is enriched in the hospital-epidemic clade and determined that it is active for DNA modification activity and significantly impacts HGT. Overall, this work is important because it provides a mechanism for the observed clade structure of E. faecium as well as a mechanism for facilitated gene exchange among hospital-epidemic E. faecium.

microbiology↗

bioRxiv2018

FUS controls the processing of snoRNAs into smaller RNA fragments that can regulate gene expression

FUS is a multifunctional protein involved in many steps of RNA metabolism, including transcription, splicing, miRNA processing and replication-dependent histone gene expression. In this paper, we show for the first time that FUS binds and negatively regulates the levels of a subset of snoRNAs in cells. Scanning of available human small RNA databases revealed the existence of smaller RNA fragments that can be processed from FUS-dependent snoRNAs. Therefore, we suggest that FUS mediates the biogenesis of snoRNA-derived small RNAs, called sdRNAs. Further in silico approaches enabled us to predict putative targets of selected FUS-dependent sdRNAs. Our results indicate that sdRNAs may bind to different regions of target mRNAs as well as to noncoding transcripts and influence the posttranscriptional level or translation of these targets. SIGNIFICANCE STATEMENTRNA metabolism is orchestrated by a complex network of RNA-protein interactions and involves various classes of RNA molecules. Small nucleolar RNAs (snoRNAs) are commonly considered essential components of the ribosome biogenesis pathway. However, recent studies have revealed that snoRNAs can also be fragmented into small entities called snoRNA-derived RNAs (sdRNAs), which have been linked to multiple cancer types and thus may serve as next-generation prognostic or diagnostic biomarkers. In this paper, a multifunctional protein, FUS, was shown to be involved in the biogenesis of snoRNA-derived fragments. Furthermore, we combined bioinformatic analyses with complementary experimental approaches to elucidate the role of FUS-dependent sdRNAs in gene expression regulation. Our findings reveal the considerable regulatory potential of this new class of small noncoding RNAs.

molecular biology↗

bioRxiv2018

Genetic Determinants of Cortical Structure (Thickness, Surface Area and Volumes) among Disease Free Adults in the CHARGE Consortium

Cortical thickness, surface area and volumes (MRI cortical measures) vary with age and cognitive function, and in neurological and psychiatric diseases. We examined heritability, genetic correlations and genome-wide associations of cortical measures across the whole cortex, and in 34 anatomically predefined regions. Our discovery sample comprised 22,822 individuals from 20 cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the United Kingdom Biobank. Significant associations were replicated in the Enhancing Neuroimaging Genetics through Meta-analysis (ENIGMA) consortium, and their biological implications explored using bioinformatic annotation and pathway analyses. We identified genetic heterogeneity between cortical measures and brain regions, and 161 genome-wide significant associations pointing to wnt/{beta}-catenin, TGF-{beta} and sonic hedgehog pathways. There was enrichment for genes involved in anthropometric traits, hindbrain development, vascular and neurodegenerative disease and psychiatric conditions. These data are a rich resource for studies of the biological mechanisms behind cortical development and aging.

genetics↗

bioRxiv2018

The functional circular RNA, ciRS-7 (CDR1as), is biosynthesized using back-splicing promoted by inverted mammalian-wide MIRs but not primate-specific Alus

Circular RNAs (circRNAs) are stable noncoding RNAs with a closed circular structure. One of the first and best studied circRNAs is ciRS-7 (CDR1as) that acts as a regulator of the microRNA miR-7, however, the biosynthesis pathway has remained an enigma. Here we delineate the biosynthesis pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. ciRS-7 gene lacks these elements but, instead, we identified a set of flanking inverted elements belonging to the mammalian-wide interspersed repeat (MIR) family. Splicing reporter assays in HEK293 cells demonstrated that these inverted MIRs are required to generate ciRS-7 through a back-splicing and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatics searches, we identified several other MIR-dependent circRNAs that we confirmed experimentally. We propose that MIR-mediated RNA circularization constitutes a new widespread biosynthesis principle for mammalian circRNAs.

molecular biology↗

bioRxiv2018

Genome-wide identification and expression specificity analysis of the DNA methyltransferase gene family under adversity stresses in cotton

DNA methylation is an important epigenetic mode of genomic DNA modification that is an important part of maintaining epigenetic content and regulating gene expression. DNA methyltransferases (MTases) are the key enzymes in the process of DNA methylation. Thus far, there has been no systematic analysis the DNA MTases found in cotton. In this study, the whole genome of cotton C5-Mtase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome. In this study, 51 DNA MTase genes were identified, of which 8 belonged to G. raimondii (group D), 9 belonged to G. arboretum L. (group A), 16 belonged to G. hirsutum L. (group AD1) and 18 belonged to G. barbadebse L. (group AD2). Systematic evolutionary analysis divided the 51 genes into four subfamilies, including 7 MET homologous proteins, 25 CMT homologous proteins, 14 DRM homologous proteins and 5 DNMT2 homologous proteins. Further studies showed that the DNA MTases in cotton were more phylogenetically conserved. The comparison of their protein domains showed that the C-terminal functional domain of the 51 proteins had six conserved motifs involved in methylation modification, indicating that the protein has a basic catalytic methylation function and the difference in the N-terminal regulatory domains of the 51 proteins divided the proteins into four classes, MET, CMT, DRM and DNMT2, in which DNMT2 lacks an N-terminal regulatory domain. Gene expression in cotton is not the same under different stress treatments. Different expression patterns of DNA MTases show the functional diversity of the cotton DNA methyltransferase gene family. VIGS silenced Gossypium hirsutum l. in the cotton seedling of DNMT2 family gene GhDMT6, after stress treatment the growth condition was better than the control. The distribution of DNA MTases varies among cotton species. Different DNA MTase family members have different genetic structures, and the expression level changes with different stresses, showing tissue specificity. Under salt and drought stress, G. hirsutum L. TM-1 increased the number of genes more than G. raimondii and G. arboreum L. Shixiya 1. The resistance of Gossypium hirsutum L.TM-1 to cold, drought and salt stress was increased after the plants were silenced with GhDMT6 gene.

genomics↗

bioRxiv2018

Evolution of the highly repetitive PEVK region of titin across mammals

The protein titin plays a key role in vertebrate muscle where it acts like a giant molecular spring. Despite its importance and conservation over vertebrate evolution, a lack of high quality annotations in non-model species makes comparative evolutionary studies of titin challenging. The PEVK region of titin--named for its high proportion of Pro-Glu-Val-Lys amino acids--is particularly difficult to annotate due to its abundance of alternatively spliced isoforms and short, highly repetitive exons. To understand PEVK evolution across mammals, we first developed a bioinformatics tool, PEVK_Finder, to annotate PEVK exons from genomic sequences of titin and then applied it to a diverse set of mammals. PEVK_Finder consistently outperforms standard annotation tools across a broad range of conditions and improves annotations of the PEVK region in non-model mammalian species. We find that the PEVK region can be divided into two subregions (PEVK-N, PEVK-C) with distinct patterns of evolutionary constraint and divergence. The bipartite nature of the PEVK region has implications for titin diversification. In the PEVK-N region, certain exons are conserved and may be essential, but natural selection also acts on particular codons. This region is also rich in glutamate and may contribute to actin binding. In the PEVK-C, exons are more homogenous and length variation of the PEVK region may provide the raw material for evolutionary adaptation in titin function. Taken together, we find that the very complexity that makes titin a challenge for annotation tools may also promote evolutionary adaptation.

genomics↗

bioRxiv2018

Cross-talk between the receptor tyrosine kinases AXL and ERBB3 regulates invadopodia formation in melanoma cells

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix, and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified novel invadopodia regulators, the knock-down of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances invadopodia formation and function (e.g., ABL2, AXL, CSK). Particularly intriguing was the discovery that the receptor tyrosine kinase AXL displays a dual regulatory function, manifested by enhancement of invadopodia function upon knock-down or long-term inhibition, as well as following its over-expression. We show here that this apparent contradiction may be attributed to the capacity of AXL to directly stimulate invadopodia; yet its suppression up-regulates the ERBB3 signaling pathway, which consequently activates core invadopodia regulators, and greatly enhances invadopodia function. Bioinformatic analysis of multiple melanoma cells points to an inverse expression pattern of AXL and ERBB3, with the apparent association of high-AXL melanomas, with high expression of invadopodia components and an invasive phenotype. The relevance of these results to melanoma metastasis in vivo, and to potential anti-invasion therapy, is discussed.

cancer biology↗

bioRxiv2018

Pediatric Brainstem Encephalitis Outbreak Investigation with Metagenomic Next-Generation Sequencing

In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF). RNA was extracted from CSF (n=20), plasma (n=9), stool (n=15) and nasopharyngeal samples (n=16) from 10 children with brainstem encephalitis or encephalomyelitis and 10 contemporaneous pediatric controls with presumed viral meningitis or encephalitis. Unbiased complementary DNA libraries were sequenced, and microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Metagenomic next-generation sequencing (mNGS) was concordant with qRT-PCR for all samples positive by PCR (n=25). In virus-negative samples (n=35), mNGS detected virus in 28.6% (n=10), including 5 CSF samples. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, mNGS increased the proportion of EV-positive samples from 42% (25/60) to 57% (34/60) (McNemars test; p-value = 0.0077). For CSF, mNGS doubled the number of pathogen-positive samples (McNemars test; p-value = 0.074). Using phylogenetic analysis, the outbreak EV-A71 clustered with a neuroinvasive German EV-A71 isolate. Brainstem encephalitis specific, non-synonymous EV-A71 single nucleotide variants were not identified. mNGS demonstrated 100% concordance with clinical qRT-PCR of EV-related brainstem encephalitis and significantly increased the detection of enteroviruses. Our findings increase the probability that neurologic complications observed were virus-induced rather than para-infectious. A comprehensive genomic analysis confirmed that the EV-A71 outbreak strain was closely related to a neuroinvasive German EV-A71 isolate. There were no clear-cut viral genomic differences that discriminated between patients with differing neurologic phenotypes.

microbiology↗

bioRxiv2018

RNAseq dataset describing transcriptional changes in cervical sensory ganglia after bilateral pyramidotomy and forelimb intramuscular gene therapy with AAV1 encoding human neurotrophin-3

Unilateral or bilateral corticospinal tract injury in the pyramids of adult rats causes changes in proprioceptive axon terminal arborization in the cervical spinal cord accompanied by hyperreflexia and abnormal movements including spasms [1, 2]. Treatment of affected forelimb muscles with an Adeno-Associated Viral Vector (AAV) encoding human neurotrophin-3 (NT3) normalizes many of these anatomical, neurophysiological and behavioural changes [1]. Interestingly, in several studies, neurotrophin-3 protein accumulates in cervical dorsal root ganglia (DRG) on the side ipsilateral to AAV injection [1, 3]. We hypothesize that neurotrophin-3 induces these changes (in proprioceptive axon wiring, proprioceptive reflex neurophysiology and sensorimotor behaviors involving proprioception) by modifying gene expression in affected cervical dorsal root ganglia (DRG). As a first step in testing this hypothesis, we analyzed the transcriptomes of cervical DRGs obtained during a previous study from naive rats and from rats after bilateral pyramidotomy (bPYX) with unilateral intramuscular injections of either AAV1-CMV-NT3 or AAV1-CMV-EGFP made 24h after injury [1]. Ten weeks after surgery, Poly(A) RNAs and small RNAs from C6 to C8 DRGs on the treated side were sequenced. We detected mRNAs or small RNAs that were significantly regulated under three conditions (bPYX+GFP vs naive; bPYX+NT3 versus naive; bPYX+NT3 vs bPYX+GFP). We identified mRNAs and small RNAs whose expression level was altered after pyramidotomy and normalized by neurotrophin-3 treatment. A bioinformatic analysis enabled us to identify genes that are likely to be expressed in proprioceptors after injury and which were regulated by neurotrophin-3 in the direction expected from other datasets involving knockout or overexpression of neurotrophin-3. This dataset will help us and others identify genes in sensory neurons whose expression levels are regulated by neurotrophin-3 treatment. This may help identify novel therapeutic targets to improve sensation and movement after neurological injury. Data has been deposited in the Gene Expression Omnibus (GSE82197).

neuroscience↗

bioRxiv2018

Quantitative multi-locus metabarcoding and waggle dance interpretation reveal honey bee spring foraging patterns in Midwest agroecosystems

We explored the pollen foraging behavior of honey bee colonies situated in the corn and soybean dominated agroecosystems of central Ohio over a month-long period using both pollen metabarcoding and waggle dance inference of spatial foraging patterns. For molecular pollen analysis we developed simple and cost-effective laboratory and bioinformatics methods. Targeting four plant barcode loci (ITS2, rbcL, trnL and trnH), we implemented metabarcoding library preparation and dual-indexing protocols designed to minimize amplification biases and index mis-tagging events. We constructed comprehensive, curated reference databases for hierarchical taxonomic classification of metabarcoding data and used these databases to train the Metaxa2 DNA sequence classifier. Comparisons between morphological and molecular palynology provide strong support for the quantitative potential of multi-locus metabarcoding. Results revealed consistent foraging habits between locations and show clear trends in the phenological progression of honey bee spring foraging in these agricultural areas. Our data suggest that three key taxa, woody Rosaceae such as pome fruits and hawthorns, Salix, and Trifolium provided the majority of pollen nutrition during the study. Spatially, these foraging patterns were associated with a significant preference for forests and tree lines relative to crop fields and herbaceous land cover.

ecology↗

bioRxiv2018

Beating Swords into Ploughshares: Domestication of a Phage Lysin for Housekeeping Function

Temperate phages constitute a potentially beneficial genetic reservoir for bacterial innovation despite being selfish entities encoding an infection cycle inherently at odds with bacterial fitness. These phages integrate their genomes into the bacterial host during infection, donating new, but deleterious, genetic material: the phage genome encodes toxic genes, such as lysins, that kill the bacterium during the phage infection cycle. Remarkably, some bacteria have exploited the destructive properties of phage genes for their own benefit by co-opting them as toxins for functions related to bacterial warfare, virulence, and secretion. However, do toxic phage genes ever become raw material for functional innovation? Here we report on a toxic phage gene whose product has lost its toxicity and has become a domain of a core cellular factor, SpmX, throughout the bacterial order Caulobacterales. Using a combination of phylogenetics, bioinformatics, structural biology, cell biology, and biochemistry, we have investigated the origin and function of SpmX and determined that its occurrence is the result of the detoxification of a phage peptidoglycan hydrolase gene. We show that the retained, attenuated activity of the phage-derived domain plays an important role in proper cell morphology and developmental regulation in representatives of this large bacterial clade. To our knowledge, this is the first observation of phage gene domestication in which a toxic phage gene has been co-opted for a housekeeping function.

microbiology↗

bioRxiv2018

PolyA tracks and poly-lysine repeats are the Achilles heel of Plasmodium falciparum

Plasmodium falciparum, the causative agent of human malaria, is an apicomplexan parasite with a complex, multi-host life cycle. Sixty percent of transcripts from its extreme AT-rich (81%) genome possess coding polyadenosine (polyA) runs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate that transcripts with polyA runs encoding poly-lysine are hot spots for ribosome stalling and frameshifting, eliciting mRNA surveillance pathways and attenuating protein synthesis in the majority of prokaryotic and eukaryotic organisms. Here, we show that the P. falciparum translational machinery is paradigm-breaking. Using bioinformatic and biochemical approaches, we demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately transcribed and translated in P. falciparum cells. Translation of polyA tracks in the parasite does not elicit any response from mRNA surveillance pathways usually seen in host human cells or organisms with similar AT content. The translation efficiency and accuracy of the parasite protein synthesis machinery reveals a unique role of ribosomes in the evolution and adaptation of P. falciparum to an AU-rich transcriptome and polybasic amino sequences. Finally, we show that the ability of P. falciparum to synthesize long poly-lysine repeats has given this parasite a unique protein exportome and an advantage in infectivity that can be suppressed by addition of exogenous poly-basic polymers.

microbiology↗

bioRxiv2018

Genomic sequence capture of haemosporidian parasites: Methods and prospects for enhanced study of host-parasite evolution

Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus, and Leucocytoozoon) represent an exciting multi-host, multi-parasite system in ecology and evolution. Global research in this field accelerated after 1) the publication in 2000 of PCR protocols to sequence a haemosporidian mitochondrial (mtDNA) barcode, and 2) the development in 2009 of an open-access database to document the geographic and host ranges of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from bird hosts, however, has been technically challenging, slowing the transition to genomic-scale sequencing techniques. We extend a recently-developed sequence capture method to obtain hundreds of haemosporidian nuclear loci from wild bird samples, which typically have low levels of infection, or parasitemia. We tested 51 infected birds from Peru and New Mexico and evaluated locus recovery in light of variation in parasitemia, divergence from reference sequences, and pooling strategies. Our method was successful for samples with parasitemia as low as [~]0.03% (3 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one Leucocytozoon sample), and using the most cost-effective pooling strategy tested. Phylogenetic relationships estimated with >300 nuclear loci were well resolved, providing substantial improvement over the mtDNA barcode. We provide protocols for sample preparation and sequence capture including custom probe kit sequences, and describe our bioinformatics pipeline using aTRAM 2.0, PHYLUCE, and custom Perl and Python scripts. This approach can be applied to the tens of thousands of avian samples that have already been screened for haemosporidians, and greatly improve our understanding of parasite speciation, biogeography, and evolutionary dynamics.

genomics↗

bioRxiv2018

Sexual selection rewires reproductive protein networks

Polyandry drives postcopulatory sexual selection (PCSS), resulting in rapid evolution of male ejaculate traits. Critical to male and female fitness, the ejaculate is known to contain rapidly evolving seminal fluid proteins (SFPs) produced by specialized male secretory accessory glands. The evidence that rapid evolution of some SFPs is driven by PCSS, however, is indirect, based on either plastic responses to changes in the sexual selection environment or correlative macroevolutionary patterns. Moreover, such studies focus on SFPs that represent but a small component of the accessory gland proteome. Neither how SFPs function with other reproductive proteins, nor how PCSS influences the underlying secretory tissue adaptations and content of the accessory gland, has been addressed at the level of the proteome. Here we directly test the hypothesis that PCSS results in rapid evolution of the entire male accessory gland proteome and protein networks by taking a system-level approach, combining divergent experimental evolution of PCSS in Drosophila pseudoobscura (Dpse), high resolution mass spectrometry (MS) and proteomic discovery, bioinformatics and population genetic analyses. We demonstrate that PCSS influences the abundance of over 200 accessory gland proteins, including SFPs. A small but significant number of these proteins display molecular signatures of positive selection. Divergent PCSS also results in fundamental and remarkably compartmentalized evolution of accessory gland protein networks in which males subjected to strong PCSS invest in protein networks that serve to increase protein production whereas males subjected to relaxed PCSS alters protein networks involved in protein surveillance and quality. These results directly demonstrate that PCSS is a key evolutionary driver that shapes not only individual reproductive proteins, but rewires entire reproductive protein networks.\n\nThe abbreviations used are

evolutionary biology↗