scholarly journals Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing

Cell Reports ◽  
2021 ◽  
Vol 37 (7) ◽  
pp. 110022
Author(s):  
Szi Kay Leung ◽  
Aaron R. Jeffries ◽  
Isabel Castanho ◽  
Ben T. Jordan ◽  
Karen Moore ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Alternative Splicing ◽  
Full Length ◽  
Isoform Diversity ◽  
Mouse Cerebral Cortex ◽  
Full Length Transcript ◽  
Human And Mouse

scholarly journals Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

2020 ◽  
Author(s):  
A.R. Jeffries ◽  
SK. Leung ◽  
I. Castanho ◽  
K. Moore ◽  
J.P. Davies ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cerebral Cortex ◽  
Alternative Splicing ◽  
Intron Retention ◽  
Full Length ◽  
True Nature ◽  
Isoform Diversity ◽  
Transcript Diversity ◽  
Human And Mouse

AbstractAlternative splicing is a post-transcriptional regulatory mechanism producing multiple distinct mRNA molecules from a single pre-mRNA. Alternative splicing has a prominent role in the central nervous system, impacting neurodevelopment and various neuronal functions as well as being increasingly implicated in brain disorders including autism, schizophrenia and Alzheimer’s disease. Standard short-read RNA-Seq approaches only sequence fragments of the mRNA molecule, making it difficult to accurately characterize the true nature of RNA isoform diversity. In this study, we used long-read isoform sequencing (Iso-Seq) to generate full-length cDNA sequences and map transcript diversity in the human and mouse cerebral cortex. We identify widespread RNA isoform diversity amongst expressed genes in the cortex, including many novel transcripts not present in existing genome annotations. Alternative splicing events were found to make a major contribution to RNA isoform diversity in the cortex, with intron retention being a relatively common event associated with nonsense-mediated decay and reduced transcript expression. Of note, we found evidence for transcription from novel (unannotated genes) and fusion events between neighbouring genes. Although global patterns of RNA isoform diversity were found to be generally similar between human and mouse cortex, we identified some notable exceptions. We also identified striking developmental changes in transcript diversity, with differential transcript usage between human adult and fetal cerebral cortex. Finally, we found evidence for extensive isoform diversity in genes associated with autism, schizophrenia and Alzheimer’s disease. Our data confirm the importance of alternative splicing in the cerebral cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide this transcript level data as a resource to the scientific community.

scholarly journals CELF2 regulates the species-specific alternative splicing of TREM2

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.

Abstract 905: Alternative Splicing of HMGCR is Associated with Plasma LDL Cholesterol Response to Simvastatin

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Marisa W Medina ◽  
Feng Gao ◽  
Weiming Ruan ◽  
Dana Crawford ◽  
Jerome I Rotter ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Ldl Cholesterol ◽  
Residual Activity ◽  
Full Length ◽  
Nucleotide Polymorphisms ◽  
Response Distribution ◽  
Response Status ◽  
Full Length Transcript

Statins can substantially lower plasma LDL cholesterol and reduce risk for coronary heart disease, but their efficacy varies among individuals. To test whether this variation is related to single nucleotide polymorphisms (SNPs) in the gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase ( HMGCR ), the key regulatory enzyme of cholesterol synthesis and the target of statin inhibition, we resequenced HMGCR exons, splice sites and 1.5kb of 5′ and 3′ flanking regions in 96 individuals. Subjects were equally derived from the upper and the lower 5% of the LDL-response distribution from the Cholesterol and Pharmacogenetics (CAP) study, a 6 week trial in which 944 subjects were treated with 40 mg/day simvastatin. Intronic SNP 20144 (rs3846662) was significantly (p<0.05) associated with high versus low LDL cholesterol response status, and was part of a previously reported haplotype associated with a 14.8% smaller reduction in LDL cholesterol in the CAP cohort. Since SNP 20144 is adjacent to exon 13, a site of HMGCR alternative splicing, we measured expression levels of the splice variant lacking exon 13 ( HMGCRv 1 ) in 170 immortalized lymphocytes derived from the CAP subjects, and found that the SNP was associated with greater statin-induced HMGCR alternative splicing (p<0.05). Moreover, there was a significant correlation between the increased magnitude of expression of HMGCRv 1 following statin exposure in vitro and smaller reductions of total cholesterol, LDL cholesterol, apoB and triglycerides in the plasma of the corresponding subjects in vivo (p<0.0001). There was no relationship between the full-length transcript and in vivo response. Additionally, we used siRNA to specifically knock-down expression of the full-length HMGCR transcript by 68%, resulting in cells enriched in the HMGCRv 1 transcript. We then measured HMGCR enzyme activity and found that the remaining enzyme demonstrated 9 –15% greater residual activity in the presence of simvastatin concentrations ranging from 0.15nM to 6.0nM (n=10). These results suggest that HMGCR genetic variation influences the production of an HMGCR isoform with reduced statin sensitivity, and that variation in expression of this isoform is a determinant of inter-individual differences in statin response.

EPCO-14. MULTIFACETED TRANSCRIPTOMIC AND PROTEOMIC ANALYSES IDENTIFIED PUTATIVE ALTERNATIVE SPLICING-DERIVED CELL SURFACE ANTIGENS IN GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi4-vi4
Author(s):  
Takahide Nejo ◽  
Darwin Kwok ◽  
Kevin Leung ◽  
Lin Wang ◽  
Albert Wang ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Surface ◽  
Surface Antigens ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Validation Dataset ◽  
Sequencing Data ◽  
Cell Surface Antigens ◽  
Proteomic Analyses ◽  
Full Length Transcript

Abstract BACKGROUND To develop effective immunotherapy for gliomas, it is crucial to expand the repertoire of targetable antigens. Recent studies have suggested that alternative splicing (AS), or its deriving tumor-specific junctions (“neojunctions”), could generate cryptic amino acid sequences that can be a source of neoantigens. In this study, we investigated neojunctions based on multifaceted transcriptomic and proteomic analyses, seeking the potential cell surface antigens that may be targeted by CAR. METHODS For screening, we analyzed bulk RNA-sequencing data of TCGA-GBM/LGG with high tumor purity (n = 429) and GTEx normal tissues (n = 9,166). Cohorts of spatially mapped intratumoral samples and longitudinally collected tumors were used to determine clonality and stability of the candidate neojunctions. Nanopore long-read amplicon sequencing was deployed to confirm the full-length transcript sequence. Their protein-level expression was explored by analyzing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM proteomics dataset. RESULTS In the screening analysis comparing TCGA and GTEx datasets, we identified 218 neojunctions with adequate expression, prevalence, and tumor-specificity. Of these, 12 were predicted to be cell-surface antigens. Eight of the 12, such as BCAN, DLL3, and PTPRZ1, were also observed in multiple cases of another validation dataset. In the analysis of tumors with spatially mapped intratumoral samples, 7 of the 12 were recurrently detected in no less than 50% of the samples in multiple cases. In addition, 5 of the 12 were found to be conserved in primary and recurrent pairs of tumors in multiple cases. Full-length transcript sequencing corroborated our predictions based on short reads, and also demonstrated more complex AS patterns. Finally, CPTAC-GBM proteomics analysis identified one cryptic peptide that substantiated the corresponding transcriptome-based prediction. CONCLUSION: We identified neojunctions with the potential to generate cell-surface antigens. These multifaceted transcriptomic and proteomic analyses provide the rationale to pursue the development of immunotherapy targeting neojunction-derived antigens.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Molecule ◽  
High Fat Diet ◽  
Human Heart ◽  
Full Length ◽  
Funding Source ◽  
Smrt Sequencing ◽  
High Fat ◽  
Exon 1 ◽  
Novel Exon

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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