Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.

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Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

Scientific Reports ◽

10.1038/srep39094 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Anne-Laure Bougé ◽

Eva Murauer ◽

Emmanuelle Beyne ◽

Julie Miro ◽

Jessica Varilh ◽

...

Keyword(s):

Skeletal Muscle ◽

Alternative Splicing ◽

Cdna Sequence ◽

454 Sequencing ◽

Exon Skipping ◽

Rna Seq ◽

Splicing Pattern ◽

Alternative Splicing Events ◽

Diagnostic Research ◽

Dmd Gene

Abstract We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

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Strategies for Identifying RNA Splicing Regulatory Motifs and Predicting Alternative Splicing Events

PLoS Computational Biology ◽

10.1371/journal.pcbi.0040021 ◽

2008 ◽

Vol 4 (1) ◽

pp. e21 ◽

Cited By ~ 13

Author(s):

Dirk Holste ◽

Uwe Ohler

Keyword(s):

Alternative Splicing ◽

Rna Splicing ◽

Regulatory Motifs ◽

Alternative Splicing Events

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Sequence and Evolutionary Features for the Alternatively Spliced Exons of Eukaryotic Genes

International Journal of Molecular Sciences ◽

10.3390/ijms20153834 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3834 ◽

Cited By ~ 3

Author(s):

Shi-Yi Chen ◽

Cao Li ◽

Xianbo Jia ◽

Song-Jia Lai

Keyword(s):

Alternative Splicing ◽

High Throughput Sequencing ◽

Point Of View ◽

Sequencing Technologies ◽

Eukaryotic Genes ◽

Splicing Pattern ◽

Evolutionary Features ◽

Alternatively Spliced ◽

Alternative Splicing Events ◽

Alternatively Spliced Exons

Alternative splicing of pre-mRNAs is a crucial mechanism for maintaining protein diversity in eukaryotes without requiring a considerable increase of genes in the number. Due to rapid advances in high-throughput sequencing technologies and computational algorithms, it is anticipated that alternative splicing events will be more intensively studied to address different kinds of biological questions. The occurrences of alternative splicing mean that all exons could be classified to be either constitutively or alternatively spliced depending on whether they are virtually included into all mature mRNAs. From an evolutionary point of view, therefore, the alternatively spliced exons would have been associated with distinctive biological characteristics in comparison with constitutively spliced exons. In this paper, we first outline the representative types of alternative splicing events and exon classification, and then review sequence and evolutionary features for the alternatively spliced exons. The main purpose is to facilitate understanding of the biological implications of alternative splicing in eukaryotes. This knowledge is also helpful to establish computational approaches for predicting the splicing pattern of exons.

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Extensive Changes in Transcription Dynamics Reflected on Alternative Splicing Events in Systemic Lupus Erythematosus Patients

Genes ◽

10.3390/genes12081260 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1260

Author(s):

Sofia Papanikolaou ◽

George K. Bertsias ◽

Christoforos Nikolaou

Keyword(s):

Systemic Lupus Erythematosus ◽

Alternative Splicing ◽

Lupus Erythematosus ◽

Rna Splicing ◽

Intron Retention ◽

Mrna Translation ◽

Sequencing Data ◽

Systemic Lupus ◽

Alternative Splicing Events ◽

Transcriptional Output

In addition to increasing the complexity of the transcriptional output, alternative RNA splicing can lead to the reduction of mRNA translation or the production of non-functional or malfunctional proteins, thus representing a vital component of the gene regulation process. Herein, we set out to detect and characterize alternative splicing events that occur in whole-blood samples of patients with Systemic Lupus Erythematosus (SLE) as compared to healthy counterparts. Through the implementation of a computational pipeline on published RNA-sequencing data, we identified extensive changes in the transcription dynamics affecting a large number of genes. We found a predominance of intron retention events, with the majority introducing premature stop codons, suggestive of gene repression, in both inactive and active SLE patient samples. Alternative splicing affected a distinct set of genes from the ones detected as differentially expressed in the same comparisons, while alternatively spliced genes tended to reside in genome areas associated with increased gene co-expression. Functional analysis of genes affected by alternative splicing pointed towards particular functions related to metabolism and histone acetylation as of potential interest. Together, our findings underline the importance of incorporating alternative splicing analyses in the context of molecular characterization of complex diseases such as SLE.

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IMMU-02. NEOANTIGENS ARISING FROM ALTERNATIVE SPLICING EVENTS MAY BE TARGETED BY TUMOR INFILTRATING LYMPHOCYTES IN GLIOBLASTOMAS

Neuro-Oncology ◽

10.1093/neuonc/noz175.496 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi118-vi119

Author(s):

Alexander Lee ◽

Yang Pan ◽

Aaron Mochizuki ◽

Mildred Galvez ◽

Frances Chow ◽

...

Keyword(s):

T Cells ◽

Alternative Splicing ◽

Single Cell ◽

Rna Splicing ◽

Ex Vivo ◽

Tumor Infiltrating Lymphocytes ◽

Sequencing Data ◽

Synonymous Mutations ◽

Alternative Splicing Events ◽

Infiltrating Lymphocytes

Abstract INTRODUCTION Alternative splicing, the cellular process that converts premature mRNA to mature mRNA and allows for single genes to produce multiple protein products, is frequently dysregulated in many cancers, including glioblastoma. However, along with non-synonymous mutations in the DNA, altered splicing mechanisms in cancers may produce novel antigens (so-called neoantigens) that distinguish cancer cells from healthy cells and can thus be targeted by the immune system. METHODS We developed a new computation pipeline (IRIS – Isoform peptides from RNA splicing for Immunotherapy targets Screening) that took bulk RNA-sequencing data from 23 glioblastoma patient tumor samples and predicted neoantigens that may arise from alternative splicing events. We prioritized predicted neoantigens that arose in HLA*A02:01 and HLA*A03:01 patients and selected 8 potential neoantigens to generate peptide:MHC Class 1 dextramers. We tested PBMCs and/or ex vivo expanded tumor infiltrating lymphocytes (TIL) from 6 of our glioblastoma patients against these dextramers, sorted for any neoantigen-reactive T cells, and performed single-cell RNAsequencing on the sorted population to determine the TCR sequence. RESULTS Among the 8 predicted neoantigens tested, 7 of the neoantigens were recognized by at least 1 patient’s T cells. 1 HLA*A03:01 epitope was recognized in 3 of the 4 HLA*A03:01 patients tested and this epitope was highly positive in an expanded TIL population, representing 1.7% of all CD3+ CD8+ cells. When we sorted for those neoantigen reactive T cells from the expanded TIL population and performed single-cell RNAsequencing, we found 325 unique T cell clonotypes, but the top 10 clonotypes represented 83.6% of all TCR clonotypes. The most frequent TCR clonotype represented 39.1% of the repertoire and suggests that clonal expansion of a select few TCR clones occurred within the tumor. CONCLUSIONS In total, our data indicates that neoantigens arising from alternative splicing events may represent a potential target for immunotherapy in glioblastoma.

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RNA splicing alterations induce a cellular stress response associated with poor prognosis in AML

10.1101/2020.01.10.895714 ◽

2020 ◽

Cited By ~ 1

Author(s):

Govardhan Anande ◽

Nandan P. Deshpande ◽

Sylvain Mareschal ◽

Aarif M. N. Batcha ◽

Henry R. Hampton ◽

...

Keyword(s):

Alternative Splicing ◽

Stress Response ◽

Rna Splicing ◽

Protein Translation ◽

Good Prognosis ◽

Cellular Stress Response ◽

Splicing Factor ◽

Machine Learning Techniques ◽

Molecular Feature ◽

Alternative Splicing Events

RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are relatively uncommon in Acute Myeloid Leukemia (AML, < 20%). We examined whether RNA splicing differences exist in AML even in the absence of splicing factor mutations. Analyzing RNA-seq data from two independent cohorts of AML patients, we identified recurrent differential alternative splicing between patients with poor and good prognosis. These alternative splicing events occurred even in patients without any discernible splicing factor mutations. The alternative splicing events recurrently occurred in genes involved in specific molecular functions, primarily related to protein translation. Developing informatics tools to predict the functional impact of alternative splicing on the translated protein, we discovered that ~45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced in patients, and the splicing of their target transcripts were also altered. By studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and up- regulation of inflammation-related genes. Lastly, using machine learning techniques, we identified a set of four genes whose alternative splicing can refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance.

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