[PDF] Novel Regulation Of The Nonsense Mediated Rna Decay Pathway eBook

Author : Andrew Nickless
Publisher :
Page : 205 pages
File Size : 24,32 MB
Release : 2016
Category : Electronic dissertations
ISBN :

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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 : Lynne E. Maquat
Publisher :
Page : 277 pages
File Size : 34,54 MB
Release : 2006
Category : ARN
ISBN : 9780429090189

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Nonsense-Mediated mRNA Decay is the first book devoted to nonsense-mediated mRNA decay (NMD) The rationale for such a book is the enormous information that studies of NMD have provided on the intricacies of post-transcriptional gene expression. The first five sections of the book are divided according to organism and begin with chapters on S. cerevisiae and mammals, from which most NMD data derive. Chapters within these sections discuss the two basic ways cells differentiate between a termination codon that elicits NMD and one that does not. Nonsense-Mediated mRNA Decay makes clear that studies of NMD provide a unique opportunity to examine the coupling of mRNA translation and decay, and relationships between mRNP structure and function. The importance of NMD is evident from descriptions of its influence on the expression of genes that are essential for many cellular processes, including cell differentiation, cell maintenance and cell death. This book is, essentially, meant to be a one-stop source of information that fuels the fires of future experimentation.

Author : Lynne E. Maquat
Publisher : CRC Press
Page : 277 pages
File Size : 23,95 MB
Release : 2006-02-28
Category : Science
ISBN : 9781587062957

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Nonsense-Mediated mRNA Decay is the first book devoted to nonsense-mediated mRNA decay (NMD). The rationale for such a book is the enormous information that studies of NMD have provided on the intricacies of post-transcriptional gene expression. The first five sections of the book are divided according to organism and begin with chapters on S. cerevisiae and mammals, from which most NMD data derive. Chapters within these sections discuss the two basic ways cells differentiate between a termination codon that elicits NMD and one that does not. Nonsense-Mediated mRNA Decay makes clear that studies of NMD provide a unique opportunity to examine the coupling of mRNA translation and decay, and relationships between mRNP structure and function. The importance of NMD is evident from descriptions of its influence on the expression of genes that are essential for many cellular processes, including cell differentiation, cell maintenance and cell death. This book is, essentially, meant to be a one-stop source of information that fuels the fires of future experimentation.

Author : Lynne E. Maquat
Publisher : Academic Press
Page : 463 pages
File Size : 25,56 MB
Release : 2011-09-02
Category : Science
ISBN : 0080923321

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Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, and RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Messenger RNA (mRNA) decay is now emerging as an important control point and a major contributor to gene expression. Continuing identification of the protein factors and cofactors and mRNA instability elements responsible for mRNA decay allow researchers to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation. Covers the nonsense-mediated mRNA decay (NMD) or mRNA surveillance pathway Expert researchers introduce the most advanced technologies and techniques Offers step-by-step lab instructions, including necessary equipment and reagents

Author : Alexander De Jong Robertson
Publisher :
Page : 187 pages
File Size : 33,75 MB
Release : 2014
Category :
ISBN :

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Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA and protein are controlled by the proteins' inherent affinities for different RNA sequences as well as other features such as translation and RNA structure which affect the accessibility of mRNA. The stabilities of mRNA transcripts are regulated by nonsense-mediated mRNA decay (NMD), a quality control degradation pathway. In this thesis, I present a novel method for high throughput characterization of the binding affinities of proteins for mRNA sequences and an integrative analysis of NMD using deep sequencing data. This thesis describes RNA Bind-n-Seq (RBNS), which comprehensively characterizes the sequence and structural specificity of RNA binding proteins (RBPs), and application to the developmentally-regulated splicing factors RBFOX2, MBNL1 and CELF1/CUGBP1. For each factor, the canonical motifs are recovered as well as additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in UGC-containing motifs. In a project investigating how NMD shapes the embryonic transcriptome, this thesis presents integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). Over 200 direct UPF1 binding targets are identified using crosslinking/immunoprecipitation-sequencing (CLIP-seq). Results from ribosome foot printing show that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression.

Author : Abigael Cheruiyot
Publisher :
Page : 169 pages
File Size : 18,16 MB
Release : 2020
Category : Electronic dissertations
ISBN :

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Nonsense mediated RNA decay (NMD) is an RNA surveillance pathway present in all eukaryotes that detects and degrades nonsense mRNAs, which contain pre-mature translation termination codons. Nonsense mRNAs are prevalent when pre-mRNA splicing is altered or defective. Interestingly, defective pre-mRNA splicing is emerging as a major driver of cancer development, including development of myelodysplastic syndrome (MDS), leukemia, and some solid tumors. Moreover, pre-mRNA splicing is also thought to enhance NMD in human cells, although it's still unclear whether and how splicing or splicing factors promote NMD. The role of NMD in regulating mis-spliced mRNA and the link between NMD and RNA splicing, suggest that understanding the process of NMD in the context of normal and defective splicing may hold some clues on developing therapies to treat cancers with dysregulated splicing. To better understand the process of NMD, we have developed a novel NMD reporter system to measure NMD activity in individual human cells and used it to perform a genome-wide CRISPR/Cas9 KO screen to identify genes that promote NMD. We found that the SF3B spliceosome complex promotes NMD without splicing of the target mRNA, suggesting that recruitment of certain spliceosome factors, but not pre-mRNA splicing per se, promotes NMD. In the context of defective splicing, we found that expression of cancer-associated spliceosome mutants (including mutant SF3B1) attenuate NMD. Importantly, cancer cells harboring spliceosome mutations were remarkably sensitive to inhibition of NMD. Therefore, inhibition of NMD is a novel potential therapeutic strategy to treat cancers with defective splicing. This finding suggests that small molecule inhibitors of NMD are needed to facilitate development of therapies that target the NMD pathway. In this dissertation, we have evaluated the use of two different compounds to inhibit NMD. SMG1i directly targets SMG1, the only kinase in the NMD pathway, while Compound C, a commonly used AMPK inhibitor, inhibits NMD indirectly probably by down-regulating NMD factors. Compound C is, however, non-specific, but its derivatives may generate specific NMD inhibitors. Collectively, our studies shed some new light on the process of NMD in the context of normal or defective splicing, uncover NMD as a novel vulnerability of cancers with defective splicing, and provide promising lead compounds for developing therapies that target NMD for cancer treatment.

Author : Courtney Elizabeth French
Publisher :
Page : 86 pages
File Size : 36,18 MB
Release : 2016
Category :
ISBN :

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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.