Project description:Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both transcription and degradation determines the amount, timing, and location of protein translation. This balance is of particular importance in neurons, which are among the most metabolically active and morphologically complex cells in the body. As a result, any disruptions in RNA degradation can have dramatic consequences for neuronal health. In this chapter, we will first discuss mechanisms of RNA stabilization and decay. We will then explore how the disruption of these pathways can lead to neurodegenerative disease.
Project description:The process of RNA splicing is fundamental in contributing to proteomic diversity and regulating gene expression. Dysregulation of splicing is associated with various human disorders, including cancer. Through functional studies, this study sought to examine the potential impact of seven variants within six inherited cancer-related genes on RNA splicing patterns in Turkish cancer patients. Upon detecting variants using Next-Generation Sequencing (NGS), we used Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Sanger sequencing to elucidate the effects of these variants on splicing. Three of the seven variants demonstrated no discernible effect on RNA, while four exhibited pathogenic characteristics. Specifically, the variants APC c.532-1G>A rs1554072547, BRCA1c.4358-3A>G rs1567779966, BRCA2c.7436-1G>C rs81002830 and MSH3c.1897-1G>A rs1744149615 were identified as pathogenic, while the variants BLMc.4076+4T>G rs183176301, RB1c.2489+2T>C rs1555294636 and RB1c.1050-2A>G rs? were found to be benign from a splicing perspective. These findings highlight the importance of verifying the precise consequences of splice-site variants through experimental analysis, given their potential implications for genetic disorders and cancer predisposition. This research contributes to the understanding of splice-site variants in inherited cancer predisposition, particularly among Turkish cancer patients. It emphasizes the necessity for further exploration into the mechanisms and functional consequences of alternative splicing for potential therapeutic interventions in cancer.
Project description:The success of checkpoint inhibitors in cancer therapy is largely attributed to activating the patient's immune response to their tumor's neoantigens arising from DNA mutations. This realization has motivated the interest in personal cancer vaccines based on sequencing the patient's tumor DNA to discover neoantigens. Here we propose an additional, unrecognized source of tumor neoantigens. We show that errors in transcription of microsatellites (MS) and mis-splicing of exons create highly immunogenic frameshift (FS) neoantigens in tumors. The sequence of these FS neoantigens are predictable, allowing creation of a peptide array representing all possible neoantigen FS peptides. This array can be used to detect the antibody response in a patient to the FS peptides. A survey of 5 types of cancers reveals peptides that are personally reactive for each patient. This source of neoantigens and the method to discover them may be useful in developing cancer vaccines.
Project description:A-to-I RNA editing diversifies human transcriptome to confer its functional effects on the downstream genes or regulations, potentially involving in neurodegenerative pathogenesis. Its variabilities are attributed to multiple regulators, including the key factor of genetic variants. To comprehensively investigate the potentials of neurodegenerative disease-susceptibility variants from the view of A-to-I RNA editing, we analyzed matched genetic and transcriptomic data of 1596 samples across nine brain tissues and whole blood from two large consortiums, Accelerating Medicines Partnership-Alzheimer's Disease and Parkinson's Progression Markers Initiative. The large-scale and genome-wide identification of 95 198 RNA editing quantitative trait loci revealed the preferred genetic effects on adjacent editing events. Furthermore, to explore the underlying mechanisms of the genetic controls of A-to-I RNA editing, several top RNA-binding proteins were pointed out, such as EIF4A3, U2AF2, NOP58, FBL, NOP56 and DHX9, since their regulations on multiple RNA-editing events were probably interfered by these genetic variants. Moreover, these variants may also contribute to the variability of other molecular phenotypes associated with RNA editing, including the functions of 3 proteins, expressions of 277 genes and splicing of 449 events. All the analyses results shown in NeuroEdQTL (https://relab.xidian.edu.cn/NeuroEdQTL/) constituted a unique resource for the understanding of neurodegenerative pathogenesis from genotypes to phenotypes related to A-to-I RNA editing.
Project description:Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.
Project description:Alternative splicing of pre-mRNA increases genetic diversity, and recent studies estimate that most human multiexon genes are alternatively spliced. If this process is not highly regulated and accurate, it leads to mis-splicing events, which may result in proteins with altered function. A growing body of work has implicated mis-splicing events in a range of diseases, including cancer, neurodegenerative diseases, and muscular dystrophies. Understanding the mechanisms that cause aberrant splicing events and how this leads to disease is vital for designing effective therapeutic strategies. In this review, we focus on advances in therapies targeting splicing, and highlight the animal models developed to recapitulate disease phenotypes as a model for testing these therapies.
Project description:Ubiquitous in eukaryotes, circular RNAs (circRNAs) comprise a large class of mostly non-coding RNAs produced by back-splicing. Although some circRNAs have demonstrated biochemical activities, whether most circRNAs are functional is unknown. Here, we test the hypothesis that circRNA production primarily results from splicing error and so is deleterious instead of beneficial. In support of the error hypothesis, our analysis of RNA sequencing data from 11 shared tissues of humans, macaques, and mice finds that (1) back-splicing is much rarer than linear-splicing, (2) the rate of back-splicing diminishes with the splicing amount, (3) the overall prevalence of back-splicing in a species declines with its effective population size, and (4) circRNAs are overall evolutionarily unconserved. We estimate that more than 97% of the observed circRNA production is deleterious. We identify a small number of functional circRNA candidates, and the genome-wide trend strongly suggests that circRNAs are largely non-functional products of splicing errors.
Project description:Alexander disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander disease.
Project description:BackgroundRNA-Seq has emerged as the standard for measuring gene expression and is an important technique often used in studies of human disease. Gene expression quantification involves comparison of the sequenced reads to a known genomic or transcriptomic reference. The accuracy of that quantification relies on there being enough unique information in the reads to enable bioinformatics tools to accurately assign the reads to the correct gene.ResultsWe apply 12 common methods to estimate gene expression from RNA-Seq data and show that there are hundreds of genes whose expression is underestimated by one or more of those methods. Many of these genes have been implicated in human disease, and we describe their roles. We go on to propose a two-stage analysis of RNA-Seq data in which multi-mapped or ambiguous reads can instead be uniquely assigned to groups of genes. We apply this method to a recently published mouse cancer study, and demonstrate that we can extract relevant biological signal from data that would otherwise have been discarded.ConclusionsFor hundreds of genes in the human genome, RNA-Seq is unable to measure expression accurately. These genes are enriched for gene families, and many of them have been implicated in human disease. We show that it is possible to use data that may otherwise have been discarded to measure group-level expression, and that such data contains biologically relevant information.
Project description:RNA was purified from fusiform gyrus tissue sections of autopsy-confirmed Alzheimer's cases and neurologically normal age-matched controls. The "SAM.ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0007261