Target-capture full-length double-strand cDNA sequencing for alternative splicing analysis

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

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Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20176301013 ◽

2017 ◽

Vol 63 (1) ◽

pp. 13-26 ◽

Cited By ~ 5

Author(s):

D.D. Zhdanov ◽

D.A. Vasina ◽

E.V. Orlova ◽

V.S. Orlova ◽

V.S. Pokrovsky ◽

...

Keyword(s):

T Cells ◽

Alternative Splicing ◽

Malignant Transformation ◽

Cd4 T Cells ◽

Replicative Senescence ◽

Telomerase Activity ◽

Full Length ◽

Leukemic Cells ◽

Proliferative Potential ◽

Human Telomerase Reverse Transcriptase

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

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The full-length transcriptome of Spartina alterniflora reveals the complexity of high salt tolerance in monocotyledonous halophyte

10.1101/680819 ◽

2019 ◽

Cited By ~ 2

Author(s):

Wenbin Ye ◽

Taotao Wang ◽

Wei Wei ◽

Shuaitong Lou ◽

Faxiu Lan ◽

...

Keyword(s):

Gene Expression ◽

Salt Stress ◽

Alternative Splicing ◽

Salt Tolerance ◽

Protein Kinases ◽

Spartina Alterniflora ◽

High Salt ◽

Full Length ◽

Salt Tolerant ◽

High Salt Tolerance

ABSTRACTSpartina alterniflora (Spartina) is the only halophyte in the salt marsh. However, the molecular basis of its high salt tolerance remains elusive. In this study, we used PacBio full-length single molecule long-read sequencing and RNA-seq to elucidate the transcriptome dynamics of high salt tolerance in Spartina by salt-gradient experiments (0, 350, 500 and 800 mM NaCl). We systematically analyzed the gene expression diversity and deciphered possible roles of ion transporters, protein kinases and photosynthesis in salt tolerance. Moreover, the co-expression network analysis revealed several hub genes in salt stress regulatory networks, including protein kinases such as SaOST1, SaCIPK10 and three SaLRRs. Furthermore, high salt stress affected the gene expression of photosynthesis through down-regulation at the transcription level and alternative splicing at the post-transcriptional level. In addition, overexpression of two Spartina salt-tolerant genes SaHSP70-I and SaAF2 in Arabidopsis significantly promoted the salt tolerance of transgenic lines. Finally, we built the SAPacBio website for visualizing the full-length transcriptome sequences, transcription factors, ncRNAs, salt-tolerant genes, and alternative splicing events in Spartina. Overall, this study sheds light on the high salt tolerance mechanisms of monocotyledonous-halophyte and demonstrates the potential of Spartina genes for engineering salt-tolerant plants.

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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.

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Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis)

Scientific Reports ◽

10.1038/s41598-019-39286-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 15

Author(s):

Dahe Qiao ◽

Chun Yang ◽

Juan Chen ◽

Yan Guo ◽

Yan Li ◽

...

Keyword(s):

Alternative Splicing ◽

Secondary Metabolism ◽

Camellia Sinensis ◽

Tea Plant ◽

Full Length

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CELF2 regulates the species-specific alternative splicing of TREM2

Scientific Reports ◽

10.1038/s41598-020-75057-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Motoaki Yanaizu ◽

Chika Washizu ◽

Nobuyuki Nukina ◽

Jun-ichi Satoh ◽

Yoshihiro Kino

Keyword(s):

Alternative Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Full Length ◽

Splicing Regulation ◽

Nonsense Mediated Mrna Decay ◽

Specific Alternative ◽

Species Specific ◽

Human And Mouse ◽

Paralogous Proteins

Abstract Genetic variations of TREM2 have been implicated as a risk factor of Alzheimer’s disease (AD). Recent studies suggest that the loss of TREM2 function compromises microglial responses to the accumulation of amyloid beta. Previously, we found that exon 3 of TREM2 is an alternative exon whose skipping leads to a reduction in full-length TREM2 protein by inducing nonsense-mediated mRNA decay. Here, we aimed to identify factors regulating TREM2 splicing. Using a panel of RNA-binding proteins, we found that exon 3 skipping of TREM2 was promoted by two paralogous proteins, CELF1 and CELF2, which were both linked previously with risk loci of AD. Although the overexpression of both CELF1 and CELF2 enhanced exon 3 skipping, only CELF2 reduced the expression of full-length TREM2 protein. Notably, the TREM2 ortholog in the green monkey, but not in the mouse, showed alternative splicing of exon 3 like human TREM2. Similarly, splicing regulation of exon 3 by CELF1/2 was found to be common to humans and monkeys. Using chimeric minigenes of human and mouse TREM2, we mapped a CELF-responsive sequence within intron 3 of human TREM2. Collectively, our results revealed a novel regulatory factor of TREM2 expression and highlighted a species-dependent difference of its regulation.

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When small RNAs become smaller: non - canonical functions of snoRNAs and their derivatives

Acta Biochimica Polonica ◽

10.18388/abp.2016_1330 ◽

2017 ◽

Vol 63 (4) ◽

Cited By ~ 14

Author(s):

Anna Maria Mleczko ◽

Kamilla Bąkowska-Żywicka

Keyword(s):

Alternative Splicing ◽

Small Rnas ◽

Metabolic Stress ◽

Full Length ◽

Human Diseases ◽

Cajal Bodies ◽

Prader Willi Syndrome ◽

Small Nucleolar Rnas ◽

Stress Regulation ◽

Nucleolar Rnas

Small nucleolar RNAs (snoRNAs) are molecules placed in the cell nucleolus and in Cajal bodies. Many scientific reports clearly show that snoRNAs are not only responsible for modifications of other RNAs but also possess multiple other functions such as metabolic stress regulation or modulation of alternative splicing. Full-length snoRNAs as well as small RNAs derived from snoRNAs have been implied in human diseases such as cancer or Prader – Willi Syndrome. In this review we would like to describe these non – canonical roles of snoRNAs and their derivatives with the emphasis on their role in human diseases.

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