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

2008 ◽  
Vol 4 (1) ◽  
pp. e21 ◽  
Author(s):  
Dirk Holste ◽  
Uwe Ohler
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Regulatory Motifs ◽  
Alternative Splicing Events

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

2021 ◽  
Vol 22 (10) ◽  
pp. 5154
Author(s):  
Elena Putscher ◽  
Michael Hecker ◽  
Brit Fitzner ◽  
Peter Lorenz ◽  
Uwe Klaus Zettl
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Region Of Interest ◽  
Restriction Enzymes ◽  
Genomic Region ◽  
Gateway Cloning ◽  
Splicing Pattern ◽  
Processing Step ◽  
Alternative Splicing Events ◽  
Minigene Assay

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.

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

Genes ◽  
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.

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

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.

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

2020 ◽  
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.

scholarly journals Role of Alternative Splicing in Regulating Host Response to Viral Infection

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1720
Author(s):  
Kuo-Chieh Liao ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Innate Immunity ◽  
Alternative Splicing ◽  
Immune Responses ◽  
Viral Infection ◽  
Rna Splicing ◽  
Type I ◽  
Host Immune Responses ◽  
Transcriptional Regulatory Mechanisms ◽  
Host Virus Interactions

The importance of transcriptional regulation of host genes in innate immunity against viral infection has been widely recognized. More recently, post-transcriptional regulatory mechanisms have gained appreciation as an additional and important layer of regulation to fine-tune host immune responses. Here, we review the functional significance of alternative splicing in innate immune responses to viral infection. We describe how several central components of the Type I and III interferon pathways encode spliced isoforms to regulate IFN activation and function. Additionally, the functional roles of splicing factors and modulators in antiviral immunity are discussed. Lastly, we discuss how cell death pathways are regulated by alternative splicing as well as the potential role of this regulation on host immunity and viral infection. Altogether, these studies highlight the importance of RNA splicing in regulating host–virus interactions and suggest a role in downregulating antiviral innate immunity; this may be critical to prevent pathological inflammation.

scholarly journals Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wen-juan Li ◽  
Yao-hui He ◽  
Jing-jing Yang ◽  
Guo-sheng Hu ◽  
Yi-an Lin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cell Growth ◽  
Rna Splicing ◽  
Synergistic Effects ◽  
Arginine Methylation ◽  
Type I ◽  
Prostate Cancer Cells ◽  
Cancer Cell Growth ◽  
Cancer Mutations ◽  
Substrate Spectrum

AbstractNumerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

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