Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart

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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Integrative Visual Analysis of the Effects of Alternative Splicing on Protein Domain Interaction Networks

Journal of Integrative Bioinformatics ◽

10.1515/jib-2008-101 ◽

2008 ◽

Vol 5 (2) ◽

Cited By ~ 1

Author(s):

Dorothea Emig ◽

Melissa S. Cline ◽

Karsten Klein ◽

Anne Kunert ◽

Petra Mutzel ◽

...

Keyword(s):

Alternative Splicing ◽

Protein Interactions ◽

Visual Analysis ◽

Protein Isoforms ◽

Interaction Networks ◽

Protein Domain ◽

Domain Interaction ◽

Exon Arrays ◽

Exon Expression ◽

Splicing Patterns

SummaryProteins and their interactions are essential for the functioning of all organisms and for understanding biological processes. Alternative splicing is an important molecular mechanism for increasing the protein diversity in eukaryotic cells. Splicing events that alter the protein structure and the domain composition can be responsible for the regulation of protein interactions and the functional diversity of different tissues. Discovering the occurrence of splicing events and studying protein isoforms have become feasible using Affymetrix Exon Arrays. Therefore, we have developed the versatile Cytoscape plugin DomainGraph that allows for the visual analysis of protein domain interaction networks and their integration with exon expression data. Protein domains affected by alternative splicing are highlighted and splicing patterns can be compared.

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Nonsense-associated alternative splicing as a putative reno-protective mechanism in Pkhd1cyli/Pkhd1cyli mutant mice

10.1101/2021.09.22.461297 ◽

2021 ◽

Author(s):

Chaozhe Yang ◽

Naoe Harafuji ◽

Maryanne C. Odinakashukwu ◽

Ljubica Caldovic ◽

Ravindra Boddu ◽

...

Keyword(s):

Alternative Splicing ◽

Kidney Disease ◽

Protein Isoforms ◽

Mutant Mice ◽

Chromosome 1 ◽

Recessive Mutation ◽

Protective Mechanism ◽

Biliary Disease ◽

Polycystic Kidney ◽

Cystic Liver

Autosomal recessive polycystic kidney disease (ARPKD) is a hereditary hepato-renal fibrocystic disorder and a significant genetic cause of childhood morbidity and mortality. Mutations in the Polycystic Kidney and Hepatic Disease 1 (PKHD1) gene cause all typical forms of ARPKD. Several mouse strains carrying diverse genetically engineered disruptions in the orthologous Pkhd1 gene have been generated. The current study describes a novel spontaneous mouse recessive mutation causing a cystic liver phenotype resembling the hepato-biliary disease characteristic of human ARPKD. Here we describe mapping of the cystic liver mutation to the Pkhd1 interval on Chromosome 1 and identification of a frameshift mutation within Pkhd1 exon 48 predicted to result in premature translation termination. Mice homozygous for the new mutation, symbollzed Pkhd1cyli, lack renal pathology, consistent with previously generated Pkhd1 mouse mutants that fail to recapitulate human kidney disease. We have identified a profile of alternatively spliced Pkhd1 renal transcripts that are distinct in normal versus mutant mice. The Pkhd1 transcript profile in mutant kidneys is consistent with predicted outcomes of nonsense-associated alternative splicing (NAS) and nonsense mediated decay (NMD). Overall levels of Pkhd1 transcripts in mutant mouse kidneys were reduced compared to kidneys of normal mice, and Pkhd1 encoded protein in mutant kidneys was undetectable by immunoblotting. We suggest that in Pkhd1cyli/Pkhd1cyli (cyli) mice, mutation-promoted Pkhd1 alternative splicing in the kidney yields transcripts encoding low-abundance protein isoforms lacking exon 48 encoded amino acid sequences that are sufficiently functional so as to attenuate expression of a renal cystic disease phenotype.

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DoChaP: The Domain Change Presenter

10.1101/2020.12.16.423045 ◽

2020 ◽

Author(s):

Shani T. Gal-Oz ◽

Nimrod Haiat ◽

Dana Eliyahu ◽

Guy Shani ◽

Tal Shay

Keyword(s):

Alternative Splicing ◽

Rna Splicing ◽

Protein Domains ◽

Visual Presentation ◽

Structural Effect ◽

Protein Isoforms ◽

Multiple Transcripts ◽

Genome Wide ◽

Health And Disease ◽

Specific Alternative

AbstractAlternative RNA splicing results in multiple transcripts of the same gene, possibly encoding for different protein isoforms with different protein domains and functionalities. Whereas it is possible to manually determine the effect of a specific alternative splicing event on the domain composition of a particular encoded protein, the process requires the tedious integration of several data sources; it is therefore error prone and its implementation is not feasible for genome-wide characterization of domains affected by differential splicing. To fulfill the need for an automated solution, we developed the Domain Change Presenter (DoChaP), a web server for the visualization of the exon–domain association. DoChaP visualizes all transcripts of a given gene, the domains of the proteins that they encode, and the exons encoding each domain. The visualization enables a comparison between the transcripts and between the protein isoforms they encode for. The organization and visual presentation of the information makes the structural effect of each alternative splicing event on the protein structure easily identified. To enable a study of the conservation of the exon structure, alternative splicing, and the effect of alternative splicing on protein domains, DoChaP also facilitates an inter-species comparison of domain–exon associations. DoChaP thus provides a unique and easy-to-use visualization of the exon–domain association and its conservation between transcripts and orthologous genes and will facilitate the study of the functional effects of alternative splicing in health and disease.

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Evidence for tissue-specific alternative splicing of the ovine insulin receptor gene

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200597488 ◽

1998 ◽

Vol 1998 ◽

pp. 96-96

Author(s):

P.D. McGrattan ◽

A.R.G. Wylie ◽

A.J. Bjourson

Keyword(s):

Alternative Splicing ◽

Insulin Receptor ◽

Receptor Gene ◽

Protein Isoforms ◽

Common Mechanism ◽

Mrna Transcript ◽

Insulin Receptor Gene ◽

Tissue Specific ◽

Specific Regulation ◽

Exon 11

Alternative splicing of a discrete 36 base pair segment (exon 11) of the human and rat insulin receptor leads to the formation of high and low affinity isoforms differing as much as 3-fold in affinity for insulin. Alternative splicing is a common mechanism for generating protein isoforms and is often regulated in a tissue-specific fashion (Seino & Bell, 1989; Mosthaf et al., 1990). In humans, the lower affinity (B-isoform) mRNA transcript is predominantly expressed in tissues that are important for modulating glucose homeostasis such as the liver and muscle whereas the higher affinity (A-isoform) mRNA transcript is predominantly expressed in haematopoietic tissues such as spleen. Alternative splicing of the region of the ovine insulin receptor gene encoding exon 11 has recently been demonstrated (McGrattan et al., unpublished). The objective of the present study was to establish whether tissue-specific regulation of alternative splicing of the insulin receptor gene occurs in the ruminant animal.

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Alternative splicing in endothelial cells: novel therapeutic opportunities in cancer angiogenesis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01753-1 ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Anna Di Matteo ◽

Elisa Belloni ◽

Davide Pradella ◽

Ambra Cappelletto ◽

Nina Volf ◽

...

Keyword(s):

Alternative Splicing ◽

Human Cancer ◽

Single Gene ◽

Predictive Biomarkers ◽

Protein Isoforms ◽

Therapeutic Interventions ◽

Cancer Therapies ◽

Functional Changes ◽

Splicing Regulators ◽

Multiple Protein

AbstractAlternative splicing (AS) is a pervasive molecular process generating multiple protein isoforms, from a single gene. It plays fundamental roles during development, differentiation and maintenance of tissue homeostasis, while aberrant AS is considered a hallmark of multiple diseases, including cancer. Cancer-restricted AS isoforms represent either predictive biomarkers for diagnosis/prognosis or targets for anti-cancer therapies. Here, we discuss the contribution of AS regulation in cancer angiogenesis, a complex process supporting disease development and progression. We consider AS programs acting in a specific and non-redundant manner to influence morphological and functional changes involved in cancer angiogenesis. In particular, we describe relevant AS variants or splicing regulators controlling either secreted or membrane-bound angiogenic factors, which may represent attractive targets for therapeutic interventions in human cancer.

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Neurogenesis: Regulation by Alternative Splicing and Related Posttranscriptional Processes

The Neuroscientist ◽

10.1177/1073858416678604 ◽

2016 ◽

Vol 23 (5) ◽

pp. 466-477 ◽

Cited By ~ 8

Author(s):

Enrique Lara-Pezzi ◽

Manuel Desco ◽

Alberto Gatto ◽

María Victoria Gómez-Gaviro

Keyword(s):

Alternative Splicing ◽

Protein Function ◽

Posttranscriptional Regulation ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Degradation ◽

Protein Isoforms ◽

Mammalian Brain ◽

Nonsense Mediated Decay

The complexity of the mammalian brain requires highly specialized protein function and diversity. As neurons differentiate and the neuronal circuitry is established, several mRNAs undergo alternative splicing and other posttranscriptional changes that expand the variety of protein isoforms produced. Recent advances are beginning to shed light on the molecular mechanisms that regulate isoform switching during neurogenesis and the role played by specific RNA binding proteins in this process. Neurogenesis and neuronal wiring were recently shown to also be regulated by RNA degradation through nonsense-mediated decay. An additional layer of regulatory complexity in these biological processes is the interplay between alternative splicing and long noncoding RNAs. Dysregulation of posttranscriptional regulation results in defective neuronal differentiation and/or synaptic connections that lead to neurodevelopmental and psychiatric disorders.

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Gene Expression Profiles Controlled by the Alternative Splicing Factor Nova2 in Endothelial Cells

Cells ◽

10.3390/cells8121498 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1498 ◽

Cited By ~ 3

Author(s):

Elisa Belloni ◽

Anna Di Matteo ◽

Davide Pradella ◽

Margherita Vacca ◽

Christopher D. R. Wyatt ◽

...

Keyword(s):

Alternative Splicing ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Protein Isoforms ◽

Peroxisome Proliferator ◽

Bioinformatics Analyses ◽

Peroxisome Proliferator Activated Receptor ◽

Ppar Γ ◽

Significant Enrichment ◽

Steady State Levels

Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology.

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Netrin Family: Role for Protein Isoforms in Cancer

Journal of Nucleic Acids ◽

10.1155/2019/3947123 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Caroline Suzanne Bruikman ◽

Huayu Zhang ◽

Anneli Maite Kemper ◽

Janine Maria van Gils

Keyword(s):

Cell Migration ◽

Alternative Splicing ◽

Special Interest ◽

Tumor Biology ◽

Family Members ◽

Axonal Guidance ◽

Protein Isoforms ◽

Family Role ◽

Associated Proteins

Netrins form a family of secreted and membrane-associated proteins. Netrins are involved in processes for axonal guidance, morphogenesis, and angiogenesis by regulating cell migration and survival. These processes are of special interest in tumor biology. From the netrin genes various isoforms are translated and regulated by alternative splicing. We review here the diversity of isoforms of the netrin family members and their known and potential roles in cancer.

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Influence of Intron Length on Alternative Splicing of CD44

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5930 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5930-5941 ◽

Cited By ~ 49

Author(s):

Martyn V. Bell ◽

Alison E. Cowper ◽

Marie-Paule Lefranc ◽

John I. Bell ◽

Gavin R. Screaton

Keyword(s):

Alternative Splicing ◽

Genomic Structure ◽

Single Gene ◽

Intron Length ◽

Protein Isoforms ◽

Major Influence ◽

Eukaryotic Genes ◽

Alternatively Spliced ◽

Alternatively Spliced Exons

ABSTRACT Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

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