Genotyping and lipid profiling of 601 cultivated sunflower lines reveals novel genetic determinants of oil fatty acid content.

BACKGROUND: Sunflower is an important oilseed crop domesticated in North America approximately 4000 years ago. During the last century, oil content in sunflower was under strong selection. Further improvement of oil properties achieved by modulating its fatty acid composition is one of the main directions in modern oilseed crop breeding. RESULTS: We searched for the genetic basis of fatty acid content variation by genotyping 601 inbred sunflower lines and assessing their lipid and fatty acid composition. Our genome-wide association analysis based on the genotypes for 15,483 SNPs and the concentrations of 23 fatty acids, including minor fatty acids, revealed significant genetic associations for eleven of them. Identified genomic regions included the loci involved in rare fatty acids variation on chromosomes 3 and 14, explaining up to 34.5% of the total variation of docosanoic acid (22:0) in sunflower oil. CONCLUSIONS: This is the first large scale implementation of high-throughput lipidomic profiling to sunflower germplasm characterization. This study contributes to the genetic characterization of Russian sunflower collections, which made a substantial contribution to the development of sunflower as the oilseed crop worldwide, and provides new insights into the genetic control of oil composition that can be implemented in future studies.

Alternative splicing during mammalian organ development.

Alternative splicing (AS) is pervasive in mammalian genomes, yet cross-species comparisons have been largely restricted to adult tissues and the functionality of most AS events remains unclear. We assessed AS patterns across pre- and postnatal development of seven organs in six mammals and a bird. Our analyses revealed that developmentally dynamic AS events, which are especially prevalent in the brain, are substantially more conserved than nondynamic ones. Cassette exons with increasing inclusion frequencies during development show the strongest signals of conserved and regulated AS. Newly emerged cassette exons are typically incorporated late in testis development, but those retained during evolution are predominantly brain specific. Our work suggests that an intricate interplay of programs controlling gene expression levels and AS is fundamental to organ development, especially for the brain and heart. In these regulatory networks, AS affords substantial functional diversification of genes through the generation of tissue- and time-specific isoforms from broadly expressed genes.

Murine Long Noncoding RNA Morrbid Contributes in the Regulation of NRAS Splicing in Hepatocytes In Vitro.

The coupling of alternative splicing with the nonsense-mediated decay (NMD) pathway maintains quality control of the transcriptome in eukaryotes by eliminating transcripts with premature termination codons (PTC) and fine-tunes gene expression. Long noncoding RNA (lncRNA) can regulate multiple cellular processes, including alternative splicing. Previously, murine Morrbid (myeloid RNA repressor of Bcl2l11 induced death) lncRNA was described as a locus-specific controller of the lifespan of short-living myeloid cells via transcription regulation of the apoptosis-related Bcl2l11 protein. Here, we report that murine Morrbid lncRNA in hepatocytes participates in the regulation of proto-oncogene NRAS (neuroblastoma RAS viral oncogene homolog) splicing, including the formation of the isoform with PTC. We observed a significant increase of the NRAS isoform with PTC in hepatocytes with depleted Morrbid lncRNA. We demonstrated that the NRAS isoform with PTC is degraded via the NMD pathway. This transcript is presented almost only in the nucleus and has a half-life ~four times lower than other NRAS transcripts. Additionally, in UPF1 knockdown hepatocytes (the key NMD factor), we observed a significant increase of the NRAS isoform with PTC. By a modified capture hybridization (CHART) analysis of the protein targets, we uncovered interactions of Morrbid lncRNA with the SFPQ (splicing factor proline and glutamine rich)-NONO (non-POU domain-containing octamer-binding protein) splicing complex. Finally, we propose the regulation mechanism of NRAS splicing in murine hepatocytes by alternative splicing coupled with the NMD pathway with the input of Morrbid lncRNA.

Development of a low-density panel for genomic selection of pigs in Russia.

Genomic selection is routinely used worldwide in agricultural breeding. However, in Russia, it is still not used to its full potential partially due to high genotyping costs. The use of genotypes imputed from the low-density chips (LD-chip) provides a valuable opportunity for reducing the genotyping costs. Pork production in Russia is based on the conventional 3-tier pyramid involving 3 breeds; therefore, the best option would be the development of a single LD-chip that could be used for all of them. Here, we for the first time have analyzed genomic variability in 3 breeds of Russian pigs, namely, Landrace, Duroc, and Large White and generated the LD-chip that can be used in pig breeding with the negligible loss in genotyping quality. We have demonstrated that out of the 3 methods commonly used for LD-chip construction, the block method shows the best results. The imputation quality depends strongly on the presence of close ancestors in the reference population. We have demonstrated that for the animals with both parents genotyped using high-density panels high-quality genotypes (allelic discordance rate < 0.05) could be obtained using a 300 single nucleotide polymorphism (SNP) chip, while in the absence of genotyped ancestors at least 2,000 SNP markers are required. We have shown that imputation quality varies between chromosomes, and it is lower near the chromosome ends and drops with the increase in minor allele frequency. Imputation quality of the individual SNPs correlated well across breeds. Using the same LD-chip, we were able to obtain comparable imputation quality in all 3 breeds, so it may be suggested that a single chip could be used for all of them. Our findings also suggest that the presence of markers with extremely low imputation quality is likely to be explained by wrong mapping of the markers to the chromosomal positions.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

FUS(1-359) transgenic mice as a model of ALS: pathophysiological and molecular aspects of the proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120 days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.

Cooption of heat shock regulatory system for anhydrobiosis in the sleeping chironomid Polypedilum vanderplanki.

Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.

Conservation, evolution, and regulation of splicing during prefrontal cortex development in humans, chimpanzees, and macaques.

Changes in splicing are known to affect the function and regulation of genes. We analyzed splicing events that take place during the postnatal development of the prefrontal cortex in humans, chimpanzees, and rhesus macaques based on data obtained from 168 individuals. Our study revealed that among the 38,822 quantified alternative exons, 15% are differentially spliced among species, and more than 6% splice differently at different ages. Mutations in splicing acceptor and/or donor sites might explain more than 14% of all splicing differences among species and up to 64% of high-amplitude differences. A reconstructed trans-regulatory network containing 21 RNA-binding proteins explains a further 4% of splicing variations within species. While most age-dependent splicing patterns are conserved among the three species, developmental changes in intron retention are substantially more pronounced in humans.

Transcriptome analysis reveals novel regulatory mechanisms in a genome-reduced bacterium.

The avian bacterial pathogen Mycoplasma gallisepticum is a good model for systems studies due to small genome and simplicity of regulatory pathways. In this study, we used RNA-Seq and MS-based proteomics to accurately map coding sequences, transcription start sites (TSSs) and transcript 3'-ends (T3Es). We used obtained data to investigate roles of TSSs and T3Es in stress-induced transcriptional responses. We identified 1061 TSSs at a false discovery rate of 10% and showed that almost all transcription in M. gallisepticum is initiated from classic TATAAT promoters surrounded by A/T-rich sequences. Our analysis revealed the pronounced operon structure complexity: on average, each coding operon has one internal TSS and T3Es in addition to the primary ones. Our transcriptomic approach based on the intervals between the two nearest transcript ends allowed us to identify two classes of T3Es: strong, unregulated, hairpin-containing T3Es and weak, heat shock-regulated, hairpinless T3Es. Comparing gene expression levels under different conditions revealed widespread and divergent transcription regulation in M. gallisepticum. Modeling suggested that the core promoter structure plays an important role in gene expression regulation. We have shown that the heat stress activation of cryptic promoters combined with the hairpinless T3Es suppression leads to widespread, seemingly non-functional transcription.

DNA repair in Mycoplasma gallisepticum.

BACKGROUND: DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a "minimal cell" concept. RESULTS: In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. CONCLUSIONS: Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase.

An automated stochastic approach to the identification of the protein specificity determinants and functional subfamilies.

BACKGROUND: Recent progress in sequencing and 3 D structure determination techniques stimulated development of approaches aimed at more precise annotation of proteins, that is, prediction of exact specificity to a ligand or, more broadly, to a binding partner of any kind. RESULTS: We present a method, SDPclust, for identification of protein functional subfamilies coupled with prediction of specificity-determining positions (SDPs). SDPclust predicts specificity in a phylogeny-independent stochastic manner, which allows for the correct identification of the specificity for proteins that are separated on a phylogenetic tree, but still bind the same ligand. SDPclust is implemented as a Web-server http://bioinf.fbb.msu.ru/SDPfoxWeb/ and a stand-alone Java application available from the website. CONCLUSIONS: SDPclust performs a simultaneous identification of specificity determinants and specificity groups in a statistically robust and phylogeny-independent manner.