12 research outputs found
Seed transcriptome annotation reveals enhanced expression of genes related to ros homeostasis and ethylene metabolism at alternating temperatures in wild cardoon
The association among environmental cues, ethylene response, ABA signaling, and reactive oxygen species (ROS) homeostasis in the process of seed dormancy release is nowadays well-established in many species. Alternating temperatures are recognized as one of the main environmental signals determining dormancy release, but their underlying mechanisms are scarcely known. Dry after-ripened wild cardoon achenes germinated poorly at a constant temperature of 20, 15, or 10鈼, whereas germination was stimulated by 80% at alternating temperatures of 20/10鈼. Using an RNA-Seq approach, we identified 23,640 and annotated 14,078 gene transcripts expressed in dry achenes and achenes exposed to constant or alternating temperatures. Transcriptional patterns identified in dry condition included seed reserve and response to dehydration stress genes (i.e., HSPs, peroxidases, and LEAs). At a constant temperature, we observed an upregulation of ABA biosynthesis genes (i.e., NCED9), ABA-responsive genes (i.e., ABI5 and TAP), as well as other genes previously related to physiological dormancy and inhibition of germination. However, the alternating temperatures were associated with the upregulation of ethylene metabolism (i.e., ACO1, 4, and ACS10) and signaling (i.e., EXPs) genes and ROS homeostasis regulators genes (i.e., RBOH and CAT). Accordingly, the ethylene production was twice as high at alternating than at constant temperatures. The presence in the germination medium of ethylene or ROS synthesis and signaling inhibitors reduced significantly, but not completely, germination at 20/10鈼. Conversely, the presence of methyl viologen and salicylhydroxamic acid (SHAM), a peroxidase inhibitor, partially increased germination at constant temperature. Taken together, the present study provides the first insights into the gene expression patterns and physiological response associated with dormancy release at alternating temperatures in wild cardoon (Cynara cardunculus var. sylvestris).Fil: Huarte, Hector Roberto. Consejo Nacional de Investigaciones Cient铆ficas y T茅cnicas; Argentina. Universidad Nacional de Lomas de Zamora; ArgentinaFil: Puglia, Giuseppe. D.. Consiglio Nazionale delle Ricerche; ItaliaFil: Prjibelski, Andrey D.. Academia de Ciencias de Rusia.; RusiaFil: Raccuia, Salvatore A.. Consiglio Nazionale delle Ricerche; Itali
MetaGT : A pipeline for de novo assembly of metatranscriptomes with the aid of metagenomic data
While metagenome sequencing may provide insights on the genome sequences and composition of microbial communities, metatranscriptome analysis can be useful for studying the functional activity of a microbiome. RNA-Seq data provides the possibility to determine active genes in the community and how their expression levels depend on external conditions. Although the field of metatranscriptomics is relatively young, the number of projects related to metatranscriptome analysis increases every year and the scope of its applications expands. However, there are several problems that complicate metatranscriptome analysis: complexity of microbial communities, wide dynamic range of transcriptome expression and importantly, the lack of high-quality computational methods for assembling meta-RNA sequencing data. These factors deteriorate the contiguity and completeness of metatranscriptome assemblies, therefore affecting further downstream analysis. Here we present MetaGT, a pipeline for de novo assembly of metatranscriptomes, which is based on the idea of combining both metatranscriptomic and metagenomic data sequenced from the same sample. MetaGT assembles metatranscriptomic contigs and fills in missing regions based on their alignments to metagenome assembly. This approach allows to overcome described complexities and obtain complete RNA sequences, and additionally estimate their abundances. Using various publicly available real and simulated datasets, we demonstrate that MetaGT yields significant improvement in coverage and completeness of metatranscriptome assemblies compared to existing methods that do not exploit metagenomic data. The pipeline is implemented in NextFlow and is freely available fromhttps://github.com/ablab/metaGT.Peer reviewe
Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue
Systematic assessment of long-read RNA-seq methods for transcript identification and quantification
The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis
Accurate isoform discovery with IsoQuant using long reads
Publisher Copyright: 漏 2023, The Author(s).Annotating newly sequenced genomes and determining alternative isoforms from long-read RNA data are complex and incompletely solved problems. Here we present IsoQuant鈥攁 computational tool using intron graphs that accurately reconstructs transcripts both with and without reference genome annotation. For novel transcript discovery, IsoQuant reduces the false-positive rate fivefold and 2.5-fold for Oxford Nanopore reference-based or reference-free mode, respectively. IsoQuant also improves performance for Pacific Biosciences data.Peer reviewe
Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis
Abstract The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGF尾, IL1伪, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME
Modulation of Notch Signaling at Early Stages of Differentiation of Human Induced Pluripotent Stem Cells to Dopaminergic Neurons
Elaboration of protocols for differentiation of human pluripotent stem cells to dopamine neurons is an important issue for development of cell replacement therapy for Parkinson鈥檚 disease. A number of protocols have been already developed; however, their efficiency and specificity still can be improved. Investigating the role of signaling cascades, important for neurogenesis, can help to solve this problem and to provide a deeper understanding of their role in neuronal development. Notch signaling plays an essential role in development and maintenance of the central nervous system after birth. In our study, we analyzed the effect of Notch activation and inhibition at the early stages of differentiation of human induced pluripotent stem cells to dopaminergic neurons. We found that, during the first seven days of differentiation, the cells were not sensitive to the Notch inhibition. On the contrary, activation of Notch signaling during the same time period led to significant changes and was associated with an increase in expression of genes, specific for caudal parts of the brain, a decrease of expression of genes, specific for forebrain, as well as a decrease of expression of genes, important for the formation of axons and dendrites and microtubule stabilizing proteins
Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue
Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample
Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue
Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample