New insights into transcriptome variation during cattle adipocyte adipogenesis by direct RNA sequencing
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ABSTRACT:
SUBMITTER: Peng L
PROVIDER: S-EPMC10492216 | biostudies-literature | 2023 Aug
REPOSITORIES: biostudies-literature
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Project description:Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current 'RNA-sequencing' technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00-6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.
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Project description:BACKGROUND: The current epidemic of obesity has caused a surge of interest in the study of adipose tissue formation. While major progress has been made in defining the molecular networks that control adipocyte terminal differentiation, the early steps of adipocyte development and the embryonic origin of this lineage remain largely unknown. RESULTS: Here we performed genome-wide analysis of gene expression during adipogenesis of mouse embryonic stem cells (ESCs). We then pursued comprehensive bioinformatic analyses, including de novo functional annotation and curation of the generated data within the context of biological pathways, to uncover novel biological functions associated with the early steps of adipocyte development. By combining in-depth gene regulation studies and in silico analysis of transcription factor binding site enrichment, we also provide insights into the transcriptional networks that might govern these early steps. CONCLUSIONS: This study supports several biological findings: firstly, adipocyte development in mouse ESCs is coupled to blood vessel morphogenesis and neural development, just as it is during mouse development. Secondly, the early steps of adipocyte formation involve major changes in signaling and transcriptional networks. A large proportion of the transcription factors that we uncovered in mouse ESCs are also expressed in the mouse embryonic mesenchyme and in adipose tissues, demonstrating the power of our approach to probe for genes associated with early developmental processes on a genome-wide scale. Finally, we reveal a plethora of novel candidate genes for adipocyte development and present a unique resource that can be further explored in functional assays.
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Project description:BackgroundOrganellar transcriptomes are relatively under-studied systems, with data related to full-length transcripts and posttranscriptional modifications remaining sparse. Direct RNA sequencing presents the possibility of accessing a previously unavailable layer of information pertaining to transcriptomic data, as well as circumventing the biases introduced by second-generation RNA-seq platforms. Direct long-read ONT sequencing allows for the isoform analysis of full-length transcripts and the detection of posttranscriptional modifications. However, there are still relatively few projects employing this method specifically for studying organellar transcriptomes.ResultsCandida albicans is a promising model for investigating nucleo-mitochondrial interactions. This work comprises ONT sequencing of the Candida albicans mitochondrial transcriptome along with the development of a dedicated data analysis pipeline. This approach allowed for the detection of complete transcript isoforms and posttranslational RNA modifications, as well as an analysis of C. albicans deletion mutants in genes coding for the 5' and 3' mitochondrial RNA exonucleases CaPET127 and CaDSS1. It also enabled for corrections to previous studies in terms of 3' and 5' transcript ends. A number of intermediate splicing isoforms was also discovered, along with mature and unspliced transcripts and changes in their abundances resulting from disruption of both 5' and 3' exonucleolytic processing. Multiple putative posttranscriptional modification sites have also been detected.ConclusionsThis preliminary work demonstrates the suitability of direct RNA sequencing for studying yeast mitochondrial transcriptomes in general and provides new insights into the workings of the C. albicans mitochondrial transcriptome in particular. It also provides a general roadmap for analyzing mitochondrial transcriptomic data from other organisms.
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Project description:The transcriptome profiles of the model plant Arabidopsis thaliana have been extensively studied and charcaterised under different developmental and physiological conditions. However, most of these “RNA-sequencing” datasets have been generated using the sequencing of reverse-transcribed cDNAs from mRNAs that have a relatively short read length. Here, we performed direct RNA sequencing using the latest Oxford Nanopore Technology (ONT) with unusual read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been under-estimated. The ONT direct RNA sequencing technology identified transcript isoforms at a vegetative (14 day old seedlings, stage 1.04) and a reproductive stage (stage 6.00-6) when 10% of the flowers had opened. In-house software called TrackCluster was used to determine alternative transcription initiation (ATI), possible alternative polyadenylation (APA), poly(A) length, alternative splicing (AS), and fusion transcripts. Tombo software was used to detect RNA base modifications. More than 38,500 novel transcript isoforms were identified, including six categories of fusion-transcripts which may result from differential RNA processing mechanisms. Fusion-transcripts are prone to mis-assembly by sequencing with short reads using next-generation-sequencing (NGS). These new transcript isoforms provide important additions to the annotated Arabidopsis genome. The power of ONT in detecting RNA modifications was demonstrated by characterisation of the modifications between mobile mRNAs and total mRNAs. The mobile mRNAs were enriched in m5C modifications, which is consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT direct RNA sequencing greatly enhances the identification of novel RNA transcript isoforms and RNA base modifications.
2020-08-02 | GSE144828 | GEO
Project description:Conventional bacterial genome annotation provides information about coding sequences but ignores untranslated regions and operons. However, untranslated regions contain important regulatory elements as well as targets for many regulatory factors, such as small RNAs. Operon maps are also essential for functional gene analysis. In the last decade, considerable progress has been made in the study of bacterial transcriptomes through transcriptome sequencing (RNA-seq). Given the compact nature of bacterial genomes, many challenges still cannot be resolved through short reads generated using classical RNA-seq because of fragmentation and loss of the full-length information. Direct RNA sequencing is a technology that sequences the native RNA directly without information loss or bias. Here, we employed direct RNA sequencing to annotate the Vibrio parahaemolyticus transcriptome with its full features, including transcription start sites (TSSs), transcription termination sites, and operon maps. A total of 4,103 TSSs were identified. In comparison to short-read sequencing, full-length information provided a deeper view of TSS classification, showing that most internal and antisense TSSs were actually a result of gene overlap. Sequencing the transcriptome of V. parahaemolyticus grown with bile allowed us to study the landscape of pathogenicity island Vp-PAI. Some genes in this region were reannotated, providing more accurate annotation to increase precision in their characterization. Quantitative detection of operons in V. parahaemolyticus showed high complexity in some operons, shedding light on a greater extent of regulation within the same operon. Our study using direct RNA sequencing provides a quantitative and high-resolution landscape of the V. parahaemolyticus transcriptome. IMPORTANCE Vibrio parahaemolyticus is a halophilic bacterium found in the marine environment. Outbreaks of gastroenteritis resulting from seafood poisoning by these pathogens have risen over the past 2 decades. Upon ingestion by humans-often through the consumption of raw or undercooked seafood-V. parahaemolyticus senses the host environment and expresses numerous genes, the products of which synergize to synthesize and secrete toxins that can cause acute gastroenteritis. To understand the regulation of such adaptive response, mRNA transcripts must be mapped accurately. However, due to the limitations of common sequencing methods, not all features of bacterial transcriptomes are always reported. We applied direct RNA sequencing to analyze the V. parahaemolyticus transcriptome. Mapping the full features of the transcriptome is anticipated to enhance our understanding of gene regulation in this bacterium and provides a data set for future work. Additionally, this study reveals a deeper view of a complicated transcriptome landscape, demonstrating the importance of applying such methods to other bacterial models.
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