Timing of accreting neutron stars with future X-ray instruments: towards new constraints on dense matter equation of state

The Enhanced X-ray Timing and Polarimetry (eXTP) mission is a space mission to be launched in the late 2020s that is currently in development led by China in international collaboration with European partners. Here we provide a progress report on the Czech contribution to the eXTP science. We report on our simulation results performed in Opava (Institute of Physics of the Silesian University in Opava) and Prague (Astronomical Institute of the Czech Academy of Sciences), where the advanced timing capabilities of the satellite have been assessed for bright X-ray binaries that contain an accreting neutron star (NS) and exhibit the quasi-periodic oscillations. Measurements of X-ray variability originating in oscillations of fluid in the innermost parts of the accretion region determined by general relativity, such as the radial or Lense-Thirring precession, can serve for sensitive tests enabling us to distinguish between the signatures of different viable dense matter equations of state. We have developed formulae describing non-geodesic oscillations of accreted fluid and their simplified practical forms that allow for an expeditious application of the universal relations determining the NS properties. These relations, along with our software tools for studying the propagation of light in strong gravity and neutron star models, can be used for precise modeling of the X-ray variability while focusing on properties of the intended Large Area Detector (LAD). We update the status of our program and set up an electronic repository that will provide simulation results and gradual updates as the mission specifications progress toward their final formulation.Comment: 16 pages, 6 figures, proceedings of IBWS conference, eds. Martin Jelinek and Marianna Dafcikova, 22-26 May 2023 (Karlovy Vary, Czech Republic), ISSN: 1336-033

Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

Background: Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results: Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion: We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution

Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome

Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome

GAMETOPHYTE DEFECTIVE 1, a Putative Subunit of RNases P/MRP, Is Essential for Female Gametogenesis and Male Competence in Arabidopsis

RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants

Automatic Annotation of Spatial Expression Patterns via Sparse Bayesian Factor Models

Advances in reporters for gene expression have made it possible to document and quantify expression patterns in 2D–4D. In contrast to microarrays, which provide data for many genes but averaged and/or at low resolution, images reveal the high spatial dynamics of gene expression. Developing computational methods to compare, annotate, and model gene expression based on images is imperative, considering that available data are rapidly increasing. We have developed a sparse Bayesian factor analysis model in which the observed expression diversity of among a large set of high-dimensional images is modeled by a small number of hidden common factors. We apply this approach on embryonic expression patterns from a Drosophila RNA in situ image database, and show that the automatically inferred factors provide for a meaningful decomposition and represent common co-regulation or biological functions. The low-dimensional set of factor mixing weights is further used as features by a classifier to annotate expression patterns with functional categories. On human-curated annotations, our sparse approach reaches similar or better classification of expression patterns at different developmental stages, when compared to other automatic image annotation methods using thousands of hard-to-interpret features. Our study therefore outlines a general framework for large microscopy data sets, in which both the generative model itself, as well as its application for analysis tasks such as automated annotation, can provide insight into biological questions

Non-Coding RNA Prediction and Verification in Saccharomyces cerevisiae

Non-coding RNA (ncRNA) play an important and varied role in cellular function. A significant amount of research has been devoted to computational prediction of these genes from genomic sequence, but the ability to do so has remained elusive due to a lack of apparent genomic features. In this work, thermodynamic stability of ncRNA structural elements, as summarized in a Z-score, is used to predict ncRNA in the yeast Saccharomyces cerevisiae. This analysis was coupled with comparative genomics to search for ncRNA genes on chromosome six of S. cerevisiae and S. bayanus. Sets of positive and negative control genes were evaluated to determine the efficacy of thermodynamic stability for discriminating ncRNA from background sequence. The effect of window sizes and step sizes on the sensitivity of ncRNA identification was also explored. Non-coding RNA gene candidates, common to both S. cerevisiae and S. bayanus, were verified using northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data. Four ncRNA transcripts are well supported by experimental data (RUF10, RUF11, RUF12, RUF13), while one additional putative ncRNA transcript is well supported but the data are not entirely conclusive. Six candidates appear to be structural elements in 5′ or 3′ untranslated regions of annotated protein-coding genes. This work shows that thermodynamic stability, coupled with comparative genomics, can be used to predict ncRNA with significant structural elements

Functional conservation of the Drosophila hybrid incompatibility gene Lhr

<p>Abstract</p> <p>Background</p> <p>Hybrid incompatibilities such as sterility and lethality are commonly modeled as being caused by interactions between two genes, each of which has diverged separately in one of the hybridizing lineages. The gene <it>Lethal hybrid rescue </it>(<it>Lhr</it>) encodes a rapidly evolving heterochromatin protein that causes lethality of hybrid males in crosses between <it>Drosophila melanogaster </it>females and <it>D. simulans </it>males. Previous genetic analyses showed that hybrid lethality is caused by <it>D. simulans Lhr </it>but not by <it>D. melanogaster Lhr</it>, confirming a critical prediction of asymmetry in the evolution of a hybrid incompatibility gene.</p> <p>Results</p> <p>Here we have examined the functional properties of <it>Lhr </it>orthologs from multiple Drosophila species, including interactions with other heterochromatin proteins, localization to heterochromatin, and ability to complement hybrid rescue in <it>D. melanogaster</it>/<it>D. simulans </it>hybrids. We find that these properties are conserved among most <it>Lhr </it>orthologs, including <it>Lhr </it>from <it>D. melanogaster</it>, <it>D. simulans </it>and the outgroup species <it>D. yakuba</it>.</p> <p>Conclusions</p> <p>We conclude that evolution of the hybrid lethality properties of <it>Lhr </it>between <it>D. melanogaster </it>and <it>D. simulans </it>did not involve extensive loss or gain of functions associated with protein interactions or localization to heterochromatin.</p

Global Chromatin Domain Organization of the Drosophila Genome

In eukaryotes, neighboring genes can be packaged together in specific chromatin structures that ensure their coordinated expression. Examples of such multi-gene chromatin domains are well-documented, but a global view of the chromatin organization of eukaryotic genomes is lacking. To systematically identify multi-gene chromatin domains, we constructed a compendium of genome-scale binding maps for a broad panel of chromatin-associated proteins in Drosophila melanogaster. Next, we computationally analyzed this compendium for evidence of multi-gene chromatin domains using a novel statistical segmentation algorithm. We find that at least 50% of all fly genes are organized into chromatin domains, which often consist of dozens of genes. The domains are characterized by various known and novel combinations of chromatin proteins. The genes in many of the domains are coregulated during development and tend to have similar biological functions. Furthermore, during evolution fewer chromosomal rearrangements occur inside chromatin domains than outside domains. Our results indicate that a substantial portion of the Drosophila genome is packaged into functionally coherent, multi-gene chromatin domains. This has broad mechanistic implications for gene regulation and genome evolution

Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets

Alternative splicing generates protein diversity and allows for post-transcriptional gene regulation. Estimates suggest that 10% of the genes in Caenorhabditis elegans undergo alternative splicing. We constructed a splicing-sensitive microarray to detect alternative splicing for 352 cassette exons and tested for changes in alternative splicing of these genes during development. We found that the microarray data predicted that 62/352 (∼18%) of the alternative splicing events studied show a strong change in the relative levels of the spliced isoforms (>4-fold) during development. Confirmation of the microarray data by RT-PCR was obtained for 70% of randomly selected genes tested. Among the genes with the most developmentally regulated alternatively splicing was the hnRNP F/H splicing factor homolog, W02D3.11 – now named hrpf-1. For the cassette exon of hrpf-1, the inclusion isoform comprises 65% of hrpf-1 steady state messages in embryos but only 0.1% in the first larval stage. This dramatic change in the alternative splicing of an alternative splicing factor suggests a complex cascade of splicing regulation during development. We analyzed splicing in embryos from a strain with a mutation in the splicing factor sym-2, another hnRNP F/H homolog. We found that approximately half of the genes with large alternative splicing changes between the embryo and L1 stages are regulated by sym-2 in embryos. An analysis of the role of nonsense-mediated decay in regulating steady-state alternative mRNA isoforms was performed. We found that 8% of the 352 events studied have alternative isoforms whose relative steady-state levels in embryos change more than 4-fold in a nonsense-mediated decay mutant, including hrpf-1. Strikingly, 53% of these alternative splicing events that are affected by NMD in our experiment are not obvious substrates for NMD based on the presence of premature termination codons. This suggests that the targeting of splicing factors by NMD may have downstream effects on alternative splicing regulation

Semi-supervised learning for the identification of syn-expressed genes from fused microarray and in situ image data

Background: Gene expression measurements during the development of the fly Drosophila melanogaster are routinely used to find functional modules of temporally co-expressed genes. Complimentary large data sets of in situ RNA hybridization images for different stages of the fly embryo elucidate the spatial expression patterns. Results: Using a semi-supervised approach, constrained clustering with mixture models, we can find clusters of genes exhibiting spatio-temporal similarities in expression, or syn-expression. The temporal gene expression measurements are taken as primary data for which pairwise constraints are computed in an automated fashion from raw in situ images without the need for manual annotation. We investigate the influence of these pairwise constraints in the clustering and discuss the biological relevance of our results. Conclusion: Spatial information contributes to a detailed, biological meaningful analysis of temporal gene expression data. Semi-supervised learning provides a flexible, robust and efficient framework for integrating data sources of differing quality and abundance