The dynamic landscape of fission yeast meiosis alternative-splice 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.

Download Full-text

Analysis of Transcripts and splice isoforms in Red Clover (Trifolium pratense L.) by single-molecule long-read sequencing

10.1101/330977 ◽

2018 ◽

Author(s):

Yuehui Chao ◽

Jianbo Yuan ◽

Sifeng Li ◽

Siqiao Jia ◽

Liebao Han ◽

...

Keyword(s):

Single Molecule ◽

Trifolium Pratense ◽

Intron Retention ◽

Draft Genome ◽

Red Clover ◽

Full Length ◽

Splice Isoforms ◽

Single Molecule Level ◽

Long Read ◽

Novel Isoforms

AbstractRed clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2,194 novel isoforms from novel genes were identified. A total of 5,492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8,719 including 1,86,517 transcripts have at least one poly(A) site. Furthermore, we identified 4,333 long non-coding RNAs and 3,762 fusion transcripts. Our results show the feasibility of deep sequencing full-length RNA from red clover transcriptome on a single-molecule level.

Download Full-text

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 ◽

10.1093/dnares/dsz010 ◽

2019 ◽

Vol 26 (4) ◽

pp. 301-311 ◽

Cited By ~ 11

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.

Download Full-text

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

Frontiers in Plant Science ◽

10.3389/fpls.2021.737004 ◽

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.

Download Full-text

The dynamic landscape of fission yeast meiosis alternative-splice isoforms

Genome Research ◽

10.1101/gr.208041.116 ◽

2016 ◽

Vol 27 (1) ◽

pp. 145-156 ◽

Cited By ~ 17

Author(s):

Zheng Kuang ◽

Jef D. Boeke ◽

Stefan Canzar

Keyword(s):

Fission Yeast ◽

Alternative Splice ◽

Splice Isoforms ◽

Alternative Splice Isoforms ◽

Dynamic Landscape ◽

Yeast Meiosis

Download Full-text

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

10.22541/au.160655230.05138819/v1 ◽

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.

Download Full-text

Single-molecule real-time sequencing facilitates the analysis of transcripts and splice isoforms of anthers in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

BMC Plant Biology ◽

10.1186/s12870-019-2133-z ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Chong Tan ◽

Hongxin Liu ◽

Jie Ren ◽

Xueling Ye ◽

Hui Feng ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Chinese Cabbage ◽

Anther Development ◽

Full Length ◽

Transcriptional Level ◽

Systematic Analysis ◽

Splice Isoforms ◽

A Genome ◽

Novel Isoforms

Abstract Background Anther development has been extensively studied at the transcriptional level, but a systematic analysis of full-length transcripts on a genome-wide scale has not yet been published. Here, the Pacific Biosciences (PacBio) Sequel platform and next-generation sequencing (NGS) technology were combined to generate full-length sequences and completed structures of transcripts in anthers of Chinese cabbage. Results Using single-molecule real-time sequencing (SMRT), a total of 1,098,119 circular consensus sequences (CCSs) were generated with a mean length of 2664 bp. More than 75% of the CCSs were considered full-length non-chimeric (FLNC) reads. After error correction, 725,731 high-quality FLNC reads were estimated to carry 51,501 isoforms from 19,503 loci, consisting of 38,992 novel isoforms from known genes and 3691 novel isoforms from novel genes. Of the novel isoforms, we identified 407 long non-coding RNAs (lncRNAs) and 37,549 open reading frames (ORFs). Furthermore, a total of 453,270 alternative splicing (AS) events were identified and the majority of AS models in anther were determined to be approximate exon skipping (XSKIP) events. Of the key genes regulated during anther development, AS events were mainly identified in the genes SERK1, CALS5, NEF1, and CESA1/3. Additionally, we identified 104 fusion transcripts and 5806 genes that had alternative polyadenylation (APA). Conclusions Our work demonstrated the transcriptome diversity and complexity of anther development in Chinese cabbage. The findings provide a basis for further genome annotation and transcriptome research in Chinese cabbage.

Download Full-text

Identification, evolution and alternative splicing profile analysis of Serine/Arginine-Rich Protein Splicing Factors (SR Proteins) in poplar, Arabidopsis, grape, and papaya

10.21203/rs.2.19716/v1 ◽

2019 ◽

Author(s):

Hao Wang ◽

Hanwei Yan ◽

Ting He ◽

Yamei Wang ◽

Ruijia Wang ◽

...

Keyword(s):

Alternative Splicing ◽

Profile Analysis ◽

Intron Retention ◽

Plant Stress ◽

Sr Proteins ◽

Splicing Factors ◽

Structural Domain ◽

Protein Splicing ◽

Splice Isoforms ◽

Sr Genes

Abstract Alternative splicing (AS) regulates gene expression and produces proteome diversity. Serine/Arginine-Rich Protein Splicing Factors (SR Proteins) are important splicing factors that play significant roles in spliceosome assembly and splicing regulation, and play roles in regulating plant stress. In this report, we analyzed 30 SR genes in Populus trichocarpa, 18 genes in Arabidopsis thaliana, 14 genes in Vitis vinifera and 9 genes in Carica papaya. The SR proteins contained RRM and RS conserved domains, and based on different structural domain organization were divided into six subfamilies (SR, SC, SCL, RS, RSZ and RS2Z). Gene duplication analysis revealed 94 paralogs and 408 orthologs in the four species, and the SR genes had undergone strong purifying selection. A number of stress-related cis-elements (ABRE, LTR, MBS, TC-rich repeats cis-acting element) were identified in the promoters of the SR genes. Microarray and RNA-seq data showed that SR genes expression in different tissues of the four species responded differently to abiotic stress. Poplar, Arabidopsis and grape SR genes had many splice isoforms. Moreover, 26 of 30 poplar SR genes had intron retention (IR) events, and the relative IR rates of 27 intron sites in the poplar SR genes changed significantly under cold, heat, drought and salt stress conditions. This study provides valuable resources for the gene structure, function, and evolution of poplar SR proteins.

Download Full-text

Full-length Transcriptome Analysis of Pecan (Carya illinoinensis) Kernels

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab182 ◽

2021 ◽

Author(s):

Chengcai Zhang ◽

Huadong Ren ◽

Xiaohua Yao ◽

Kailiang Wang ◽

Jun Chang

Keyword(s):

Single Molecule ◽

Molecular Mechanisms ◽

Draft Genome ◽

Alternative Polyadenylation ◽

Full Length ◽

Functional Annotations ◽

Predominant Type ◽

Long Read ◽

Non Coding Rnas ◽

Novel Isoforms

Abstract Pecan is rich in bioactive components such as fatty acids and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37 504 isoforms of 16 702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26 080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30 751, 81.99%) had functional annotations. A total of 15 465 alternative splicing (AS) events and 65 761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long non-coding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with fatty acid (FA) and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. The present study provides a full-length transcriptome dataset of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.

Download Full-text

PacBio Iso-Seq Improves the Rainbow Trout Genome Annotation and Identifies Alternative Splicing Associated With Economically Important Phenotypes

Frontiers in Genetics ◽

10.3389/fgene.2021.683408 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ali Ali ◽

Gary H. Thorgaard ◽

Mohamed Salem

Keyword(s):

Rainbow Trout ◽

Alternative Splicing ◽

Single Molecule ◽

Genome Annotation ◽

Reference Genome ◽

Cold Water ◽

Intron Retention ◽

Transcriptome Assembly ◽

Model Organism ◽

Fish Growth

Rainbow trout is an important model organism that has received concerted international efforts to study the transcriptome. For this purpose, short-read sequencing has been primarily used over the past decade. However, these sequences are too short of resolving the transcriptome complexity. This study reported a first full-length transcriptome assembly of the rainbow trout using single-molecule long-read isoform sequencing (Iso-Seq). Extensive computational approaches were used to refine and validate the reconstructed transcriptome. The study identified 10,640 high-confidence transcripts not previously annotated, in addition to 1,479 isoforms not mapped to the current Swanson reference genome. Most of the identified lncRNAs were non-coding variants of coding transcripts. The majority of genes had multiple transcript isoforms (average ∼3 isoforms/locus). Intron retention (IR) and exon skipping (ES) accounted for 56% of alternative splicing (AS) events. Iso-Seq improved the reference genome annotation, which allowed identification of characteristic AS associated with fish growth, muscle accretion, disease resistance, stress response, and fish migration. For instance, an ES in GVIN1 gene existed in fish susceptible to bacterial cold-water disease (BCWD). Besides, under five stress conditions, there was a commonly regulated exon in prolyl 4-hydroxylase subunit alpha-2 (P4HA2) gene. The reconstructed gene models and their posttranscriptional processing in rainbow trout provide invaluable resources that could be further used for future genetics and genomics studies. Additionally, the study identified characteristic transcription events associated with economically important phenotypes, which could be applied in selective breeding.

Download Full-text