scholarly journals PacBio and Illumina RNA Sequencing Identify Alternative Splicing Events in Response to Cold Stress in Two Poplar Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingli Yang ◽  
Wanqiu Lv ◽  
Liying Shao ◽  
Yanrui Fu ◽  
Haimei Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Stress Response ◽  
Cold Stress ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Intron Retention ◽  
Global Analysis ◽  
Populus Trichocarpa ◽  
Rna Seq ◽  
Long Read

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress is known in many species related to stress, but the posttranscriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of single molecular real-time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1,261 AS events in P. trichocarpa and 2,101 in P. ussuriensis among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events, which provided a new insight into the complexity and regulation of AS during cold stress response in different poplar species for the first time.

scholarly journals PacBio and Illumina RNA sequencing identify alternative splicing events in response to cold stress in two poplar species

2020 ◽  
Author(s):  
Jingli Yang ◽  
Wanqiu Lv ◽  
Minzhen Zeng ◽  
Yanrui Fu ◽  
Chenghao Li
Keyword(s):  
Alternative Splicing ◽  
Stress Response ◽  
Cold Stress ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Intron Retention ◽  
Global Analysis ◽  
Populus Trichocarpa ◽  
Rna Seq ◽  
Long Read

In eukaryotes, alternative splicing (AS) is a crucial regulatory mechanism that modulates mRNA diversity and stability. The contribution of AS to stress are known in many species related to stress. But the post-transcriptional mechanism in poplar under cold stress is still unclear. Recent studies have utilized the advantages of Single Molecular Real Time (SMRT) sequencing technology from Pacific Bioscience (PacBio) to identify full-length transcripts. We, therefore, used a combination of single-molecule long-read sequencing and Illumina RNA sequencing (RNA-Seq) for a global analysis of AS in two poplar species (Populus trichocarpa and P. ussuriensis) under cold stress. We further identified 1261 AS events in P. trichocarpa and 2101 in P. ussuriensis, among which intron retention, with a frequency of more than 30%, was the most prominent type under cold stress. RNA-Seq data analysis and annotation revealed the importance of calcium, abscisic acid, and reactive oxygen species signaling in cold stress response. Besides, the low temperature rapidly induced multiple splicing factors, transcription factors, and differentially expressed genes through AS. In P. ussuriensis, there was a rapid occurrence of AS events. This study provides new insight into the complexity and regulation of AS during cold stress response in two poplar species.

scholarly journals Direct nanopore sequencing of mRNA reveals landscape of transcript isoforms in apicomplexan parasites

2020 ◽  
Author(s):  
V Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Sequencing Platform ◽  
Apicomplexan Parasites ◽  
Oxford Nanopore ◽  
Long Read ◽  
Widespread Phenomenon

AbstractAlternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterised in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AT content of Plasmodium RNA, but also the limitations of short read sequencing in deciphering complex splicing events. In this study, we utilised the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies (ONT) to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or near full-length transcripts with comparable quantification to Illumina sequencing. By comparing this data with available gene models, we find widespread alternative splicing, particular intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This work highlights a strategy in using long read sequencing to understand splicing events at the whole transcript level, and has implications in future interpretation of RNA-seq studies.

scholarly journals The dynamic landscape of fission yeast meiosis alternative-splice isoforms

2016 ◽  
Author(s):  
Zheng Kuang ◽  
Jef D. Boeke ◽  
Stefan Canzar
Keyword(s):  
Alternative Splicing ◽  
Fission Yeast ◽  
Single Molecule ◽  
Intron Retention ◽  
Major Type ◽  
Splice Isoforms ◽  
Long Read ◽  
Dynamic Landscape ◽  
Yeast Meiosis ◽  
Novel Isoforms

AbstractAlternative splicing increases the diversity of transcriptomes and proteomes in metazoans. The extent to which alternative splicing is active and functional in unicellular organisms is less understood. Here we exploit a single-molecule long-read sequencing technique and develop an open-source software program called SpliceHunter, to characterize the transcriptome in the meiosis of fission yeast. We reveal 17017 alternative splicing events in 19741 novel isoforms at different stages of meiosis, including antisense and read-through transcripts. Intron retention is the major type of alternative splicing, followed by “alternate intron in exon”. 887 novel transcription units are detected; 60 of the predicted proteins show homology in other species and form theoretical stable structures. We compare the dynamics of novel isoforms based on the number of supporting full-length reads with those of annotated isoforms and explore the translational capacity and quality of novel isoforms. The evaluation of these factors indicates that the majority of novel isoforms are unlikely to be both condition-specific and translatable but the possibility of functional novel isoforms is not excluded. Moreover, the co-option of these unusual transcripts into newly born genes seems likely. Together, this study highlights the diversity and dynamics at the isoform level in the sexual development of fission yeast.

Large-scale analysis of the cassava transcriptome reveals the impact of cold stress on alternative splicing

2019 ◽  
Author(s):  
Shuxia Li ◽  
Xiang Yu ◽  
Zhihao Cheng ◽  
Changying Zeng ◽  
Wenbin Li ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Drought Stress ◽  
Cold Stress ◽  
Large Scale ◽  
Intron Retention ◽  
Global Analysis ◽  
Premature Termination Codon ◽  
Alternatively Spliced ◽  
Alternative Splicing Events ◽  
The Impact

Abstract Alternative splicing is an essential post-transcriptional regulatory mechanism that can impact mRNA stability and protein diversity of eukaryotic genomes. Although numerous forms of stress-responsive alternative splicing have been identified in model plants, a large-scale study of alternative splicing dynamics under abiotic stress conditions in cassava has not been conducted. Here, we report the parallel employment of isoform-Seq, ssRNA-Seq, and Degradome-Seq to investigate the diversity, abundance, and fate of alternatively spliced isoforms in response to cold and drought stress. We identified 38 164 alternative splicing events, among which 3292 and 1025 events were significantly regulated by cold and drought stress, respectively. Intron retention was the most abundant subtype of alternative splicing. Global analysis of splicing regulators revealed that the number of their alternatively spliced isoforms and the corresponding abundance were specifically modulated by cold stress. We found that 58.5% of cold-regulated alternative splicing events introduced a premature termination codon into the transcripts, and 77.6% of differential alternative splicing events were detected by Degradome-Seq. Our data reveal that cold intensely affects both quantitative and qualitative aspects of gene expression via alternative splicing pathways, and advances our understanding of the high complexity and specificity of gene regulation in response to abiotic stresses. Alternative splicing is responsible for reprogramming of the transcriptome and the sensitivity of cassava plants to cold.

scholarly journals Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
V. Vern Lee ◽  
Louise M. Judd ◽  
Aaron R. Jex ◽  
Kathryn E. Holt ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Intron Retention ◽  
Transcript Level ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Content Type ◽  
Sequencing Platform ◽  
Sequencing Studies ◽  
Long Read

ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.

scholarly journals Plant buffering against the high-light stress-induced accumulation of CsGA2ox8 transcripts via alternative splicing to finely tune gibberellin levels and maintain hypocotyl elongation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Bin Liu ◽  
Shuo Zhao ◽  
Pengli Li ◽  
Yilu Yin ◽  
Qingliang Niu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Light Intensity ◽  
Intron Retention ◽  
Light Stress ◽  
Photosynthetic Photon Flux Density ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Rna Seq ◽  
Multiple Transcripts ◽  
Novel Transcript

AbstractIn plants, alternative splicing (AS) is markedly induced in response to environmental stresses, but it is unclear why plants generate multiple transcripts under stress conditions. In this study, RNA-seq was performed to identify AS events in cucumber seedlings grown under different light intensities. We identified a novel transcript of the gibberellin (GA)-deactivating enzyme Gibberellin 2-beta-dioxygenase 8 (CsGA2ox8). Compared with canonical CsGA2ox8.1, the CsGA2ox8.2 isoform presented intron retention between the second and third exons. Functional analysis proved that the transcript of CsGA2ox8.1 but not CsGA2ox8.2 played a role in the deactivation of bioactive GAs. Moreover, expression analysis demonstrated that both transcripts were upregulated by increased light intensity, but the expression level of CsGA2ox8.1 increased slowly when the light intensity was >400 µmol·m−2·s−1 PPFD (photosynthetic photon flux density), while the CsGA2ox8.2 transcript levels increased rapidly when the light intensity was >200 µmol·m−2·s−1 PPFD. Our findings provide evidence that plants might finely tune their GA levels by buffering against the normal transcripts of CsGA2ox8 through AS.

scholarly journals The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina- and single-molecule real-time-based RNA-sequencing

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 301-311 ◽  
Author(s):  
Yue Zhang ◽  
Tonny Maraga Nyong'A ◽  
Tao Shi ◽  
Pingfang Yang
Keyword(s):  
Alternative Splicing ◽  
Real Time ◽  
Single Molecule ◽  
Intron Retention ◽  
Critical Role ◽  
Full Length ◽  
Specific Expression ◽  
Splice Isoforms ◽  
Tissue Specific ◽  
Genomic Studies

Abstract Alternative splicing (AS) plays a critical role in regulating different physiological and developmental processes in eukaryotes, by dramatically increasing the diversity of the transcriptome and the proteome. However, the saturation and complexity of AS remain unclear in lotus due to its limitation of rare obtainment of full-length multiple-splice isoforms. In this study, we apply a hybrid assembly strategy by combining single-molecule real-time sequencing and Illumina RNA-seq to get a comprehensive insight into the lotus transcriptomic landscape. We identified 211,802 high-quality full-length non-chimeric reads, with 192,690 non-redundant isoforms, and updated the lotus reference gene model. Moreover, our analysis identified a total of 104,288 AS events from 16,543 genes, with alternative 3ʹ splice-site being the predominant model, following by intron retention. By exploring tissue datasets, 370 tissue-specific AS events were identified among 12 tissues. Both the tissue-specific genes and isoforms might play important roles in tissue or organ development, and are suitable for ‘ABCE’ model partly in floral tissues. A large number of AS events and isoform variants identified in our study enhance the understanding of transcriptional diversity in lotus, and provide valuable resource for further functional genomic studies.

scholarly journals Single-Molecule Long-Read Sequencing Reveals the Diversity of Full-Length Transcripts in Leaves of Gnetum (Gnetales)

2019 ◽  
Vol 20 (24) ◽  
pp. 6350 ◽  
Author(s):  
Nan Deng ◽  
Chen Hou ◽  
Fengfeng Ma ◽  
Caixia Liu ◽  
Yuxin Tian
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Alternative Polyadenylation ◽  
Full Length ◽  
Stomatal Development ◽  
Rna Seq ◽  
Leaf Transcriptome ◽  
Long Read ◽  
Non Coding Rnas ◽  
A Site

The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.

scholarly journals Swan: a library for the analysis and visualization of long-read transcriptomes

2020 ◽  
Author(s):  
Fairlie Reese ◽  
Ali Mortazavi
Keyword(s):  
Rna Sequencing ◽  
Cell Lines ◽  
Intron Retention ◽  
Exon Skipping ◽  
Differentially Expressed ◽  
Transcript Isoforms ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

Abstract Motivation Long-read RNA-sequencing technologies such as PacBio and Oxford Nanopore have discovered an explosion of new transcript isoforms that are difficult to visually analyze using currently available tools. We introduce the Swan Python library, which is designed to analyze and visualize transcript models. Results Swan finds 4909 differentially expressed transcripts between cell lines HepG2 and HFFc6, including 279 that are differentially expressed even though the parent gene is not. Additionally, Swan discovers 285 reproducible exon skipping and 47 intron retention events not recorded in the GENCODE v29 annotation. Availability and implementation The Swan library for Python 3 is available on PyPi at https://pypi.org/project/swan-vis/ and on GitHub at https://github.com/mortazavilab/swan_vis.

scholarly journals RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 820 ◽  
Author(s):  
Chao Zeng ◽  
Michiaki Hamada
Keyword(s):  
Alternative Splicing ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Biological Diversity ◽  
Rna Binding ◽  
Regulatory Mechanism ◽  
Intron Retention ◽  
Rna Seq ◽  
Motif Analysis ◽  
Structurally Stable

Alternative splicing, a ubiquitous phenomenon in eukaryotes, is a regulatory mechanism for the biological diversity of individual genes. Most studies have focused on the effects of alternative splicing for protein synthesis. However, the transcriptome-wide influence of alternative splicing on RNA subcellular localization has rarely been studied. By analyzing RNA-seq data obtained from subcellular fractions across 13 human cell lines, we identified 8720 switching genes between the cytoplasm and the nucleus. Consistent with previous reports, intron retention was observed to be enriched in the nuclear transcript variants. Interestingly, we found that short and structurally stable introns were positively correlated with nuclear localization. Motif analysis reveals that fourteen RNA-binding protein (RBPs) are prone to be preferentially bound with such introns. To our knowledge, this is the first transcriptome-wide study to analyze and evaluate the effect of alternative splicing on RNA subcellular localization. Our findings reveal that alternative splicing plays a promising role in regulating RNA subcellular localization.

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