Selection of miRNA reference genes for plant defence studies in rice (Oryza sativa)

RT-qPCR is a widely used method to investigate the expression levels of genes under certain conditions. A key step, however, to have reliable results is the normalization of expression. For every experimental condition, suitable reference genes must be chosen. These reference genes must not be affected by differences in experimental conditions. MicroRNAs are regulatory RNA molecules, able to direct the expression levels of protein coding genes. In plants, their attributed functions range from roles in development to immunity. In this work, microRNAs (miRNAs) are evaluated for their suitability as reference genes in rice after infection with root-knot nematode Meloidogyne graminicola or after priming with beta-amino butyric acid. The evaluation was based on their amplification efficiency and their stability estimates according to geNorm, NormFinder and BestKeeper. All tested miRNAs, excluding one, were considered acceptable for normalization. Furthermore, miRNAs were validated using miRNA sequencing data. The set of microRNAs miR390-5p and miR7694-3p was found to be the most stable combination under the tested conditions. Another miRNA set consisting of miR7694-3p, miR1868 and miR1849 also shows potential to be used for miRNA expression normalization under experimental conditions beyond the scope of this study. This work is the first report on reference miRNAs in rice for the purpose of plant defence studies

MEXPRESS : visualizing expression, DNA methylation and clinical TCGA data

Background: In recent years, increasing amounts of genomic and clinical cancer data have become publically available through large-scale collaborative projects such as The Cancer Genome Atlas (TCGA). However, as long as these datasets are difficult to access and interpret, they are essentially useless for a major part of the research community and their scientific potential will not be fully realized. To address these issues we developed MEXPRESS, a straightforward and easy-to-use web tool for the integration and visualization of the expression, DNA methylation and clinical TCGA data on a single-gene level (http://mexpress.be). Results: In comparison to existing tools, MEXPRESS allows researchers to quickly visualize and interpret the different TCGA datasets and their relationships for a single gene, as demonstrated for GSTP1 in prostate adenocarcinoma. We also used MEXPRESS to reveal the differences in the DNA methylation status of the PAM50 marker gene MLPH between the breast cancer subtypes and how these differences were linked to the expression of MPLH. Conclusions: We have created a user-friendly tool for the visualization and interpretation of TCGA data, offering clinical researchers a simple way to evaluate the TCGA data for their genes or candidate biomarkers of interest

Systemic suppression of the shoot metabolism upon rice root nematode infection

Hirschmanniella oryzae is the most common plant-parasitic nematode in flooded rice cultivation systems. These migratory animals penetrate the plant roots and feed on the root cells, creating large cavities, extensive root necrosis and rotting. The objective of this study was to investigate the systemic response of the rice plant upon root infection by this nematode. RNA sequencing was applied on the above-ground parts of the rice plants at 3 and 7 days post inoculation. The data revealed significant modifications in the primary metabolism of the plant shoot, with a general suppression of for instance chlorophyll biosynthesis, the brassinosteroid pathway, and amino acid production. In the secondary metabolism, we detected a repression of the isoprenoid and shikimate pathways. These molecular changes can have dramatic consequences for the growth and yield of the rice plants, and could potentially change their susceptibility to above-ground pathogens and pests

Identification of long non-coding RNAs involved in neuronal development and intellectual disability

Recently, exome sequencing led to the identification of causal mutations in 16–31% of patients with intellectual disability (ID), leaving the underlying cause for many patients unidentified. In this context, the noncoding part of the human genome remains largely unexplored. For many long non-coding RNAs (lncRNAs) a crucial role in neurodevelopment and hence the human brain is anticipated. Here we aimed at identifying lncRNAs associated with neuronal development and ID. Therefore, we applied an integrated genomics approach, harnessing several public epigenetic datasets. We found that the presence of neuron-specific H3K4me3 confers the highest specificity for genes involved in neurodevelopment and ID. Based on the presence of this feature and GWAS hits for CNS disorders, we identified 53 candidate lncRNA genes. Extensive expression profiling on human brain samples and other tissues, followed by Gene Set Enrichment Analysis indicates that at least 24 of these lncRNAs are indeed implicated in processes such as synaptic transmission, nervous system development and neurogenesis. The bidirectional or antisense overlapping orientation relative to multiple coding genes involved in neuronal processes supports these results. In conclusion, we identified several lncRNA genes putatively involved in neurodevelopment and CNS disorders, providing a resource for functional studies

Decreasing initial telomere length in humans intergenerationally understates age-associated telomere shortening

Telomere length shortens with aging, and short telomeres have been linked to a wide variety of pathologies. Previous studies suggested a discrepancy in age-associated telomere shortening rate estimated by cross-sectional studies versus the rate measured in longitudinal studies, indicating a potential bias in cross-sectional estimates. Intergenerational changes in initial telomere length, such as that predicted by the previously described effect of a father's age at birth of his offspring (FAB), could explain the discrepancy in shortening rate measurements. We evaluated whether changes occur in initial telomere length over multiple generations in three large datasets and identified paternal birth year (PBY) as a variable that reconciles the difference between longitudinal and cross-sectional measurements. We also clarify the association between FAB and offspring telomere length, demonstrating that this effect is substantially larger than reported in the past. These results indicate the presence of a downward secular trend in telomere length at birth over generational time with potential public health implications

Underestimated effect of intragenic HIV-1 DNA methylation on viral transcription in infected individuals

Background: The HIV-1 proviral genome harbors multiple CpG islands (CpGIs), both in the promoter and intragenic regions. DNA methylation in the promoter region has been shown to be heavily involved in HIV-1 latency regulation in cultured cells. However, its exact role in proviral transcriptional regulation in infected individuals is poorly understood or characterized. Moreover, methylation at intragenic CpGIs has never been studied in depth. Results: A large, well-characterized HIV-1 patient cohort (n = 72), consisting of 17 long-term non-progressors and 8 recent seroconverters (SRCV) without combination antiretroviral therapy (cART), 15 early cART-treated, and 32 late cART-treated patients, was analyzed using a next-generation bisulfite sequencing DNA methylation method. In general, we observed low level of promoter methylation and higher levels of intragenic methylation. Additionally, SRCV showed increased promoter methylation and decreased intragenic methylation compared with the other patient groups. This data indicates that increased intragenic methylation could be involved in proviral transcriptional regulation. Conclusions: Contrasting in vitro studies, our results indicate that intragenic hypermethylation of HIV-1 proviral DNA is an underestimated factor in viral control in HIV-1-infected individuals, showing the importance of analyzing the complete proviral genome in future DNA methylation studies

Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats : a comparison between bidirectional pyrosequencing and cultivation

The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1–22.2%) and OTU (3.5–3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1–V2 and V3–V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity

PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5'-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use

Genome-wide shifts in histone modifications at early stage of rice infection with Meloidogyne graminicola

Epigenetic processes play a crucial role in the regulation of plant stress responses, but their role in plant-pathogen interactions remains poorly understood. Although histone-modifying enzymes have been observed to be deregulated in galls induced by root-knot nematodes (RKN, Meloidogyne graminicola) in rice, their influence on plant defence and their genome-wide impact has not been comprehensively investigated. First, the role of histone modifications in plant-nematode interactions was confirmed by pharmacological inhibition of histone-modifying enzymes, which all significantly affected rice susceptibility to RKN. For a more specific view, three histone marks, H3K9ac, H3K9me2, and H3K27me3, were subsequently studied by chromatin-immunoprecipitation-sequencing on RKN-induced galls at 3 days postinoculation. While levels of H3K9ac and H3K27me3 were strongly enriched, H3K9me2 was generally depleted in galls versus control root tips. Differential histone peaks were generally associated with plant defence-related genes. Transcriptome analysis using RNA-Seq and RT-qPCR-based validation revealed that genes marked with H3K9ac or H3K9me2 showed the expected activation or repression gene expression pattern, but this was not the case for H3K27me3 marks. Our results indicate that histone modifications respond dynamically to RKN infection, and that posttranslational modifications mainly at H3K9 specifically target plant defence-related genes