[PDF] Nonsense Mediated Mrna Decay Nmd In Zebrafish eBook

Author : Pooja Gangras
Publisher :
Page : pages
File Size : 41,49 MB
Release : 2019
Category : Messenger RNA.
ISBN :

GET BOOK

To study EJC function during development (Chapter 3), I generated zebrafish mutants in EJC core protein genes rbm8a and magoh. Homozygous rbm8a and magoh mutants (EJC mutants) are paralyzed and have muscle and neural defects. As expected, RNA profiling revealed that annotated aberrant and normal NMD targets are significantly upregulated in EJC mutants. An mRNA is targeted for NMD by the key NMD-regulator Upf1 when an exon-exon junction, marked by the EJC, is bound ≥ 50 nts downstream of the terminated ribosome. Surprisingly, I discovered that some upregulated normal transcripts contain a conserved proximal 3′ UTR intron (3′UI)

Author : Divine Fondzenyuy Lawir
Publisher :
Page : pages
File Size : 23,98 MB
Release : 2020
Category :
ISBN :

GET BOOK

Abstract: The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a. When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism

Author : Courtney Elizabeth French
Publisher :
Page : 86 pages
File Size : 14,29 MB
Release : 2016
Category :
ISBN :

GET BOOK

Alternative splicing plays a crucial role in increasing the amount of protein diversity and in regulating gene expression at the post-transcriptional level. In humans, almost all genes produce more than one mRNA isoform and, while the fraction varies, many other species also have a substantial number of alternatively spliced genes. Alternative splicing is regulated by splicing factors, often in a developmental time- or tissue-specific manner. Mis-regulation of alternative splicing, via mutations in splice sites, splicing regulatory elements, or splicing factors, can lead to disease states, including cancers. Thus, characterizing how alternative splicing shapes the transcriptome will lead to greater insights into the regulation of numerous cellular pathways and many aspects of human health. A critical tool for investigating alternative splicing is high-throughput mRNA sequencing (RNA-seq). This technology produces hundreds of millions of short (~100bp) sequencing reads from mRNA molecules and can be used to both discover novel transcripts and to quantify the expression of transcripts. While short read length is a limitation of the technology in its current form, RNA-seq has resulted in the discovery of hundreds of thousands of new transcripts and revealed an increased complexity of the transcriptome via alternative splicing, particularly in human. Here, I used RNA-seq analysis to investigate the global effect of post-transcriptional regulation via alternative splicing coupled to nonsense-mediated mRNA decay and to examine natural human variation in alternative splicing, particularly in genes associated with differential therapeutic drug response. The nonsense-mediated mRNA decay pathway (NMD), which degrades transcripts containing a premature termination codon, plays an important role in post-transcriptional gene regulation when coupled to alternative splicing. If a gene produces an alternative isoform that is targeted by NMD, the mRNA abundance of the protein-producing transcripts can be post-transcriptionally regulated at the alternative splicing level. This has been shown to be important in the regulation of a number of genes, including many of the splicing factors themselves. I have used RNA-seq analysis on cells where NMD has been inhibited to discover alternative isoforms that are NMD targets on a genome-wide scale in human and a number of diverse other eukaryotic species. I found that around 20% of expressed human genes are potentially regulated by alternative splicing coupled to NMD and that they fall into many different functional categories. I also found that hundreds to thousands of genes produce NMD-targeted alternative isoforms in each of frog, zebrafish, fly, fission yeast, and plant, highlighting the prevalence of this relatively under-studied method of gene regulation across the three major branches of eukaryotic organisms. I also gained insight into the features that define NMD targets, which are thought to vary between species although the field is still unclear. I find that an exon-exon junction downstream of the termination codon is a much stronger predictor of NMD than 3’ UTR length in every species except yeast. I also used RNA-seq to investigate alternative splicing in genes of pharmacologic importance. Natural human variation in the expression level and activity of genes involved in drug disposition and action (“pharmacogenes”) can affect drug response and toxicity. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. Here, we used RNA-seq to determine the expression levels and alternative splicing of 389 selected pharmacogenes across four pharmacologically relevant tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines (LCLs), which are used widely in pharmacogenomics studies. Analysis of data from 18 different individuals for each of the 5 tissues (90 samples in total) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with an independent RNA-seq dataset yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use. In conclusion, the roles of alternative splicing and NMD in the regulation of cellular processes and in human health are wide-open but critical fields of study. Advancements in sequencing technologies have had and will continue to have a huge impact on the studies of these mechanisms. New long-read technologies will likely soon be readily available and promise to greatly increase our ability to accurately interpret RNA-seq results. As the cost of sequencing continues to decrease, more and more data will be generated, allowing for a better view of how the transcriptome varies between individuals and shapes differential disease risks and drug responses.

Author : Samantha Jones
Publisher :
Page : 148 pages
File Size : 36,10 MB
Release : 2018
Category :
ISBN :

GET BOOK

The nonsense-mediated RNA decay (NMD) pathway serves as a quality control mechanism and regulator of normal gene expression. Here, we report our analysis of two gene paralogs, UPF3A and UPF3B, which we found to have opposing roles in NMD. Previous gain-of-function studies indicated that UPF3A encodes a protein exhibiting little or no NMD activity. We were therefore perplexed as to what property allowed UPF3A to survive since it arose at the dawn of vertebrates, over 400 million years ago. Using loss-of-function approaches, we found that UPF3A is a potent NMD repressor both in vitro and in vivo. We generated Upf3a-null mice and found that global UPF3A knockout causes mouse embryonic lethality and conditional knockout in male germ cells leads to spermatogenic defects, particularly at the meiotic stage where UPF3A is highly expressed. We propose that UPF3A serves as a molecular rheostat to upregulate critical NMD target mRNAs at specific developmental time points, a model supported by RNAseq analysis. In contrast to UPF3A, UPF3B does not function in the testis; instead it is critical for neural development and human cognition. Mutations in UPF3B cause a form of X-linked intellectual disability, with patients often also suffering from autism or schizophrenia. To understand UPF3B function in vivo, we generated Upf3b-null mice and found that they display defects in fear-conditioned learning and pre-pulse inhibition (PPI), a measure of sensorimotor inhibition often deficient in individuals with schizophrenia. Consistent with these defects, cortical pyramidal neurons from Upf3b-null mice exhibit decreased spine density and RNAseq analysis identified transcripts encoding key neural regulators as targets of UPF3B. Our findings demonstrated that UPF3A and UPF3B have opposing functions in the NMD pathway and act in distinct developmental processes. A major focus of my thesis work was to further investigate the role of UPF3B in neurogenesis using the olfactory system as a model. We and found (at the transcript level) fewer early OE cell types in the Upf3b-null mouse but similar numbers of mOSNs, the cells responsible for odorant detection. Pooled RNA sequencing of Upf3b-null and wild type mOSNs revealed decreased class-II olfactory receptor (olfr) expression, which may (at least partially) underlie a previously observed partial olfaction defect. When comparing pooled data with our single cell RNA sequencing (scRNAseq) results, we also found a significantly reduced number of cells expressing one or more olfrs. Closer examination of the scRNAseq data also indicated a role for UPF3B in immune response and presented the possibility that multiple OE cell types are bifunctional, playing a role in both detecting odorant molecules and responding to infection. This work was also the first to define the "translome" of pooled mOSNs, building a framework for what transcripts are more often translated in a healthy model and how that can change in a disease (in our case UPF3B-depleted) state.

Author : Andrew Nickless
Publisher :
Page : 205 pages
File Size : 12,90 MB
Release : 2016
Category : Electronic dissertations
ISBN :

GET BOOK

The nonsense mediated RNA decay (NMD) pathway maintains the integrity of cellular RNAs and controls gene expression. NMD is essential for vertebrate development and defects in NMD are associated with a variety of neurodevelopmental disorders and cancers. NMD activity is tightly regulated and is altered in response to environmental and developmental signals. To better study this dynamic pathway and to identify clinically relevant regulators of its activity, we developed a dual-color bioluminescent NMD reporter that rapidly and accurately quantifies NMD activity in mammalian cells. Using this reporter, we performed a chemical screen for small-molecule modulators of NMD activity and identified the cardiac glycosides (CGs) as potent repressors of NMD activity. Further studies on the mechanism of action of these drugs led to the finding that intracellular calcium, a key cellular signaling molecule, potently regulates NMD, with increases in intracellular calcium repressing NMD. The regulation of NMD by calcium may be exploited to treat certain genetic diseases and cancers. Regulation of NMD is particularly important to the cellular stress response. Stresses such as hypoxia, amino acid deprivation, and ER stress induce a reduction in NMD activity that promotes the expression of genes that help the cell to cope with these environmental insults. We investigated the regulation of NMD and its role in the cellular response to DNA damage and osmotic shock. We found that NMD is suppressed by persistent, but not transient, DNA damage. Conditions that constantly induce damage, such as excessive mitogenic signaling or the presence of genotoxic agents, or that prevent its swift repair, such as mutations in repair factors, generate persistent DNA lesions. Telomeres are another prominent source of persistent DNA damage because telomeric damage is difficult to repair, and telomere erosion from repeated cell divisions also elicits a protracted DNA damage response (DDR). The inhibition of NMD by persistent DNA damage is mediated in part by p38 MAP kinase signaling and augments the expression of ATF3, a stress-inducible transcription factor, by stabilizing its mRNAs. We found that osmotic shock also causes NMD inhibition but in a p38-independent manner. These results reveal a novel p38-dependent pathway that regulates NMD activity in response to persistent DNA damage which contributes to gene expression changes in damaged cells.

Author : Lam Son Nguyen
Publisher :
Page : 488 pages
File Size : 27,25 MB
Release : 2013
Category : Developmental disabilities
ISBN :

GET BOOK

Nonsense mediated mRNA decay (NMD) functions to degrade transcripts containing premature termination codons and to regulate normal expression of the transcriptome. We identified mutations in UPF3B, a core member of NMD, as the cause of intellectual disability (ID) with or without other neuro-psychiatric traits and congenital anomalies. Recently, Thrombocytopenia with Absent Radius (TAR) syndrome was linked to compound heterozygous mutations in RBM8A, another NMD factor. About 7% of TAR patients also display ID, suggesting a common etiology underlying abnormal brain development in patients with compromised NMD. To gauge into the role of NMD in the brain, we assessed the transcriptome wide deregulation as a consequence of compromised NMD in lymphoblastoid cell lines (LCLs) of patients with UPF3B mutations using RNA-Seq and exon array. We showed that up to 5% of the transcriptome was impacted on, affecting multiple genes with important neuronal functions. Among these, we demonstrated that up regulation of ARHGAP24 isoform 1, which encodes an actin cytoskeleton remodeling protein, severely disrupted the axonal growth and hindered the survival of primary hippocampal neurons. This suggested that deregulation of ARHGAP24 isoform 1, among other important neuronal genes, contributed directly to the patients' neuronal phenotype. We expanded our inquiry into the role of other NMD factors in the brain. We surveyed copy number variants (CNVs) encompassing 18 NMD genes in 57,365 patients and 20,474 controls and identified 11 de novo CNVs encompassing UPF2, another core NMD factor which encodes for a direct interacting partner of UPF3B. The transcriptome deregulation due to heterozygous loss of UPF2 in these patients was 95% similar to those of patients with UPF3B mutations, suggesting that UPF2 is a novel, dosage sensitive neuro-developmental gene. Additionally, CNVs of other four NMD genes UPF3A, SMG6, EIF4A3 and RNPS1 were also significantly enriched in the patients, and likely contributed to neurodevelopmental disease etiology. Overall, our work emphasizes the importance of properly functioning NMD in normal brain development. It also lays a solid foundation for future investigations into the causative and predisposing (when mutated) or modifying (when dosage imbalanced or polymorphic) role of NMD genes in a broad spectrum of neurological disorders.

Author :
Publisher : Academic Press
Page : 632 pages
File Size : 48,69 MB
Release : 2016-07-16
Category : Science
ISBN : 0128034882

GET BOOK

The Zebrafish: Genetics, Genomics, and Transcriptomics, Fourth Edition, is the latest volume in the Methods in Cell Biology series that looks at methods for the analysis of genetics, genomics, and transcriptomics of Zebrafish. Chapters cover such topics as gene-trap mutagenesis, genetic Screens for mutations, gene editing in zebrafish, homologous gene targeting, genome-wide RNA tomography, and developmental epigenetics and the zebrafish interactome. Covers sections on model systems and functional studies, imaging-based approaches, and emerging studies Presents chapters written by experts in the field Contains cutting-edge material on the topic

Author : J. Robert Hogg
Publisher : MDPI
Page : 107 pages
File Size : 26,79 MB
Release : 2018-07-10
Category : Science
ISBN : 3038425028

GET BOOK

This book is a printed edition of the Special Issue "Viral Interactions with Host RNA Decay Pathways" that was published in Viruses