proTRAC - a software for probabilistic piRNA cluster detection, visualization and analysis

<p>Abstract</p> <p>Background</p> <p>Throughout the metazoan lineage, typically gonadal expressed Piwi proteins and their guiding piRNAs (~26-32nt in length) form a protective mechanism of RNA interference directed against the propagation of transposable elements (TEs). Most piRNAs are generated from genomic piRNA clusters. Annotation of experimentally obtained piRNAs from small RNA/cDNA-libraries and detection of genomic piRNA clusters are crucial for a thorough understanding of the still enigmatic piRNA pathway, especially in an evolutionary context. Currently, detection of piRNA clusters relies on bioinformatics rather than detection and sequencing of primary piRNA cluster transcripts and the stringency of the methods applied in different studies differs considerably. Additionally, not all important piRNA cluster characteristics were taken into account during bioinformatic processing. Depending on the applied method this can lead to: i) an accidentally underrepresentation of TE related piRNAs, ii) overlook duplicated clusters harboring few or no single-copy loci and iii) false positive annotation of clusters that are in fact just accumulations of multi-copy loci corresponding to frequently mapped reads, but are not transcribed to piRNA precursors.</p> <p>Results</p> <p>We developed a software which detects and analyses piRNA clusters (proTRAC, probabilistic TRacking and Analysis of Clusters) based on quantifiable deviations from a hypothetical uniform distribution regarding the decisive piRNA cluster characteristics. We used piRNA sequences from human, macaque, mouse and rat to identify piRNA clusters in the respective species with proTRAC and compared the obtained results with piRNA cluster annotation from piRNABank and the results generated by different hitherto applied methods.</p> <p>proTRAC identified clusters not annotated at piRNABank and rejected annotated clusters based on the absence of important features like strand asymmetry. We further show, that proTRAC detects clusters that are passed over if a minimum number of single-copy piRNA loci are required and that proTRAC assigns more sequence reads per cluster since it does not preclude frequently mapped reads from the analysis.</p> <p>Conclusions</p> <p>With proTRAC we provide a reliable tool for detection, visualization and analysis of piRNA clusters. Detected clusters are well supported by comprehensible probabilistic parameters and retain a maximum amount of information, thus overcoming the present conflict of sensitivity and specificity in piRNA cluster detection.</p

piRNAclusterDB 2.0: update and expansion of the piRNA cluster database.

PIWI-interacting RNAs (piRNAs) and their partnering PIWI proteins defend the animal germline against transposable elements and play a crucial role in fertility. Numerous studies in the past have uncovered many additional functions of the piRNA pathway, including gene regulation, anti-viral defense, and somatic transposon repression. Further, comparative analyses across phylogenetic groups showed that the PIWI/piRNA system evolves rapidly and exhibits great evolutionary plasticity. However, the presence of so-called piRNA clusters as the major source of piRNAs is common to nearly all metazoan species. These genomic piRNA-producing loci are highly divergent across taxa and critically influence piRNA populations in different evolutionary lineages. We launched the initial version of the piRNA cluster database to facilitate research on regulation and evolution of piRNA-producing loci across tissues und species. In recent years the amount of small RNA sequencing data that was generated and the abundance of species that were studied has grown rapidly. To keep up with this recent progress, we have released a major update for the piRNA cluster database (https://www.smallrnagroup.uni-mainz.de/piRNAclusterDB), expanding it from 12 to a total of 51 species with hundreds of new datasets, and revised its overall structure to enable easy navigation through this large amount of data

Human sperm heads harbor modified YsRNA as transgenerationally inherited non-coding RNAs

Most epigenetic information is reprogrammed during gametogenesis and early development. However, some epigenetic information persists and can be inherited, a phenomenon that is common in plants. On the other hand, there are increasing examples of epigenetic inheritance in metazoans, especially for small non-coding RNAs. The presence of regulatory important RNAs in oocytes is undisputed, whereas the corresponding RNA payload in spermatozoa and its regulatory influence in the zygote and early embryogenesis is largely enigmatic. For humans, we herein describe small YRNA fragments (YsRNA) as a paternal contribution to the zygote. First, we trace the biogenesis of these YsRNAs from the source YRNAs with respect to the 5′ and 3′ modifications. Both the length and modifications make these YsRNAs reminiscent of canonical piRNAs that are not derived from piRNA clusters. Second, from the early stages of spermatogenesis to maturation in the epididymis, we observe distinct YsRNA profile dynamics in the male germline. We detected YsRNAs exclusively in mature sperm heads, the precursor of the male pronucleus in the zygote, suggesting an important role of the epididymis as a site for transmitting and modification of epigenetic information in the form of YsRNA between soma and germline in humans. Since this YsRNA-based epigenetic mechanism is effective across generations, we wondered whether this phenomenon of epigenetic inheritance has an adaptive value. Full-length YRNAs bind to Ro60, an RNA chaperone that additionally binds to non-coding RNAs. We described the profiles of non-coding RNAs bound to Ro60 in the human sperm head and detected specific binding profiles of RNA to Ro60 but no YRNA bound to Ro60. We hypothesize that the sperm head Ro60 system is functional. An adaptive phenotype mediated by the presence of a large amount of YsRNA in the sperm head, and thus as a paternal contribution in the zygote, might be related to an association of YsRNA with YRNA that prevents the adoption of a YRNA secondary structure capable of binding to Ro60. We hypothesize that preventing YRNAs from acting as Ro60-associated gatekeepers for misfolded RNAs in the zygote and early development may enhance RNA chaperoning and, thus, represent the adaptive molecular phenotype

Pinpointing the PRDM9-PRDM7 gene duplication event during primate divergence

Studies on the function of PRDM9 in model systems and its evolution during vertebrate divergence shed light on the basic molecular mechanisms of hybrid sterility and its evolutionary consequences. However, information regarding PRDM9-homolog, PRDM7, whose origin is placed in the primate evolutionary tree, as well as information about the fast-evolving DNA-binding zinc finger array of strepsirrhine PRDM9 are scarce. Thus, we aimed to narrow down the date of the duplication event leading to the emergence of PRDM7 during primate evolution by comparing the phylogenetic tree reconstructions of representative primate samples of PRDM orthologs and paralogs. To confirm our PRDM7 paralogization pattern, database-deposited sequences were used to test the presence/absence patterns expected from the paralogization timing. In addition, we extended the existing phylogenetic tree of haplorrhine PRDM9 zinc fingers with their strepsirrhine counterparts. The inclusion of strepsirrhine zinc fingers completes the PRDM9 primate phylogeny. Moreover, the updated phylogeny of PRDM9 zinc fingers showed distinct clusters of strepsirrhine, tarsier, and anthropoid degenerated zinc fingers. Here, we show that PRDM7 emerged on the branch leading to the most recent common ancestor of catarrhines; therefore, its origin is more recent than previously expected. A more detailed character evolutionary study suggests that PRDM7 may have evolved differently in Cercopithecoidea as compared to Hominoidea: it lacks the first four exons in Old World monkeys orthologs and exon 10 in Papionini orthologs. Dating the origin of PRDM7 is essential for further studies investigating why Hominoidea representatives need another putative histone methyltransferase in the testis

RDA coupled with deep sequencing detects somatic SVA-retrotranspositions and mosaicism in the human brain

Cells of the developing human brain are affected by the progressive acquisition of genetic and epigenetic alterations that have been reported to contribute to somatic mosaicism in the adult brain and are increasingly considered a possible cause of neurogenetic disorders. A recent work uncovered that the copy–paste transposable element (TE) LINE-1 (L1) is mobilized during brain development, and thus mobile non-autonomous TEs like AluY and SINE-VNTR-Alu (SVA) families can use L1 activity in trans, leading to de novo insertions that may influence the variability of neural cells at genetic and epigenetic levels. In contrast to SNPs and when considering substitutional sequence evolution, the presence or absence of TEs at orthologous loci represents highly informative clade markers that provide insights into the lineage relationships between neural cells and how the nervous system evolves in health and disease. SVAs, as the ‘youngest’ class of hominoid-specific retrotransposons preferentially found in gene- and GC-rich regions, are thought to differentially co-regulate nearby genes and exhibit a high mobility in the human germline. Therefore, we determined whether this is reflected in the somatic brain and used a subtractive and kinetic enrichment technique called representational difference analysis (RDA) coupled with deep sequencing to compare different brain regions with respect to de novo SINE-VNTR-Alu insertion patterns. As a result, we detected somatic de novo SVA integrations in all human brain regions analyzed, and the majority of de novo insertions can be attributed to lineages of telencephalon and metencephalon, since most of the examined integrations are unique to different brain regions under scrutiny. The SVA positions were used as presence/absence markers, forming informative sites that allowed us to create a maximum parsimony phylogeny of brain regions. Our results largely recapitulated the generally accepted evo-devo patterns and revealed chromosome-wide rates of de novo SVA reintegration targets and preferences for specific genomic regions, e.g., GC- and TE-rich regions as well as close proximity to genes that tend to fall into neural-specific Gene Ontology pathways. We concluded that de novo SVA insertions occur in the germline and somatic brain cells at similar target regions, suggesting that similar retrotransposition modes are effective in the germline and soma

R-loop landscape in mature human sperm: Regulatory and evolutionary implications

R-loops are three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and a displaced DNA strand. While R-loops pose a potential threat to genome integrity, they constitute 5% of the human genome. The role of R-loops in transcriptional regulation, DNA replication, and chromatin signature is becoming increasingly clear. R-loops are associated with various histone modifications, suggesting that they may modulate chromatin accessibility. To potentially harness transcription-coupled repair mechanisms in the germline, nearly the entire genome is expressed during the early stages of male gametogenesis in mammals, providing ample opportunity for the formation of a transcriptome-dependent R-loop landscape in male germ cells. In this study, our data demonstrated the presence of R-loops in fully mature human and bonobo sperm heads and their partial correspondence to transcribed regions and chromatin structure, which is massively reorganized from mainly histone to mainly protamine-packed chromatin in mature sperm. The sperm R-loop landscape resembles characteristic patterns of somatic cells. Surprisingly, we detected R-loops in both residual histone and protamine-packed chromatin and localize them to still-active retroposons, ALUs and SINE-VNTR-ALUs (SVAs), the latter has recently arisen in hominoid primates. We detected both evolutionarily conserved and species-specific localizations. Comparing our DNA-RNA immunoprecipitation (DRIP) data with published DNA methylation and histone chromatin immunoprecipitation (ChIP) data, we hypothesize that R-loops epigenetically reduce methylation of SVAs. Strikingly, we observe a strong influence of R-loops on the transcriptomes of zygotes from early developmental stages before zygotic genome activation. Overall, these findings suggest that chromatin accessibility influenced by R-loops may represent a system of inherited gene regulation

Positive selection at codon 38 of the human KCNE1 (= minK) gene and sporadic absence of 38Ser-coding mRNAs in Gly38Ser heterozygotes

<p>Abstract</p> <p>Background</p> <p>KCNE1 represents the regulatory beta-subunit of the slowly activating delayed rectifier potassium channel (IKs). Variants of KCNE1 have repeatedly been linked to the long-QT syndrome (LQTS), a disorder which predisposes to deafness, ventricular tachyarrhythmia, syncope, and sudden cardiac death.</p> <p>Results</p> <p>We here analyze the evolution of the common Gly38Ser variant (rs1805127), using genomic DNAs, complementary DNAs, and HEK293-expressed variants of altogether 19 mammalian species. The between species comparison reveals that the human-specific Gly38Ser polymorphism evolved under strong positive Darwinian selection, probably in adaptation to specific challenges in the fine-tuning of IKs channels. The involved amino acid exchanges (Asp > Gly, Gly > Ser) are moderately radical and do not induce apparent changes in posttranslational modification. According to population genetic analyses (HapMap phase II) a heterozygote advantage accounts for the maintenance of the Gly38Ser polymorphism in humans. On the other hand, the expression of the 38Ser allele seems to be disadvantageous under certain conditions, as suggested by the sporadic deficiency of 38Ser-coding mRNAs in heterozygote Central Europeans and the depletion of homozygotes 38Ser in the Yoruban sample.</p> <p>Conclusion</p> <p>We speculate that individual differences in genomic imprinting or genomic recoding might have contributed to conflicting results of recent association studies between Gly38Ser polymorphism and QT phenotype. The findings thus highlight the relevance of mRNA data in future association studies of genotypes and clinical disorders. To the best of our knowledge, they moreover provide first time evidence for a unique pattern; i.e. coincidence of positive Darwinian selection and polymorphism with a sporadically suppressed expression of one allele.</p

The genome, transcriptome, and proteome of the fish parasite Pomphorhynchus laevis (Acanthocephala)

Thorny-headed worms (Acanthocephala) are endoparasites exploiting Mandibulata (Arthropoda) and Gnathostomata (Vertebrata). Despite their world-wide occurrence and economic relevance as a pest, genome and transcriptome assemblies have not been published before. However, such data might hold clues for a sustainable control of acanthocephalans in animal production. For this reason, we present the first draft of an acanthocephalan nuclear genome, besides the mitochondrial one, using the fish parasite Pomphorhynchus laevis (Palaeacanthocephala) as a model. Additionally, we have assembled and annotated the transcriptome of this species and the proteins encoded. A hybrid assembly of long and short reads resulted in a near-complete P. laevis draft genome of ca. 260 Mb, comprising a large repetitive portion of ca. 63%. Numbers of transcripts and translated proteins (35,683) were within the range of other members of the Rotifera-Acanthocephala clade. Our data additionally demonstrate a significant reorganization of the acanthocephalan gene repertoire. Thus, more than 20% of the usually conserved metazoan genes were lacking in P. laevis. Ontology analysis of the retained genes revealed many connections to the incorporation of carotinoids. These are probably taken up via the surface together with lipids, thus accounting for the orange coloration of P. laevis. Furthermore, we found transcripts and protein sequences to be more derived in P. laevis than in rotifers from Monogononta and Bdelloidea. This was especially the case in genes involved in energy metabolism, which might reflect the acanthocephalan ability to use the scarce oxygen in the host intestine for respiration and simultaneously carry out fermentation. Increased plasticity of the gene repertoire through the integration of foreign DNA into the nuclear genome seems to be another underpinning factor of the evolutionary success of acanthocephalans. In any case, energy-related genes and their proteins may be considered as candidate targets for the acanthocephalan control

Genomics and transcriptomics of epizoic Seisonidea (Rotifera, syn. Syndermata) reveal strain formation and gradual gene loss with growing ties to the host

Background Seisonidea (also Seisonacea or Seisonidae) is a group of small animals living on marine crustaceans (Nebalia spec.) with only four species described so far. Its monophyletic origin with mostly free-living wheel animals (Monogononta, Bdelloidea) and endoparasitic thorny-headed worms (Acanthocephala) is widely accepted. However, the phylogenetic relationships inside the Rotifera-Acanthocephala clade (Rotifera sensu lato or Syndermata) are subject to ongoing debate, with consequences for our understanding of how genomes and lifestyles might have evolved. To gain new insights, we analyzed first drafts of the genome and transcriptome of the key taxon Seisonidea. Results Analyses of gDNA-Seq and mRNA-Seq data uncovered two genetically distinct lineages in Seison nebaliae Grube, 1861 off the French Channel coast. Their mitochondrial haplotypes shared only 82% sequence identity despite identical gene order. In the nuclear genome, distinct linages were reflected in different gene compactness, GC content and codon usage. The haploid nuclear genome spans ca. 46 Mb, of which 96% were reconstructed. According to ~ 23,000 SuperTranscripts, gene number in S. nebaliae should be within the range published for other members of Rotifera-Acanthocephala. Consistent with this, numbers of metazoan core orthologues and ANTP-type transcriptional regulatory genes in the S. nebaliae genome assembly were between the corresponding numbers in the other assemblies analyzed. We additionally provide evidence that a basal branching of Seisonidea within Rotifera-Acanthocephala could reflect attraction to the outgroup. Accordingly, rooting via a reconstructed ancestral sequence led to monophyletic Pararotatoria (Seisonidea+Acanthocephala) within Hemirotifera (Bdelloidea+Pararotatoria). Conclusion Matching genome/transcriptome metrics with the above phylogenetic hypothesis suggests that a haploid nuclear genome of about 50 Mb represents the plesiomorphic state for Rotifera-Acanthocephala. Smaller genome size in S. nebaliae probably results from subsequent reduction. In contrast, genome size should have increased independently in monogononts as well as bdelloid and acanthocephalan stem lines. The present data additionally indicate a decrease in gene repertoire from free-living to epizoic and endoparasitic lifestyles. Potentially, this reflects corresponding steps from the root of Rotifera-Acanthocephala via the last common ancestors of Hemirotifera and Pararotatoria to the one of Acanthocephala. Lastly, rooting via a reconstructed ancestral sequence may prove useful in phylogenetic analyses of other deep splits