Comprehensive Discovery and Analysis of RNA Binding Protein-dependent Post-transcriptional Events in Mammalian Systems PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Comprehensive Discovery and Analysis of RNA Binding Protein-dependent Post-transcriptional Events in Mammalian Systems PDF full book. Access full book title Comprehensive Discovery and Analysis of RNA Binding Protein-dependent Post-transcriptional Events in Mammalian Systems by Stephanie C. Huelga. Download full books in PDF and EPUB format.
Author: Stephanie C. Huelga
Publisher:
ISBN: 9781303990236
Category :
Languages : en
Pages : 221
Get Book Here
Book Description
Nascent transcripts produced by RNA polymerase II in eukaryotic cells are subject to extensive processing prior to the generation of a functional messenger RNA (mRNA). These RNAs are generally found coated with RNA binding proteins (RBPs), which act in concert to regulate RNA processing events, including splicing, polyadenylation, and RNA stability. The aggregate effect of various RBPs on a given RNA transcript eventually dictates its fate, a phenomenon referred to as the RNP code. The precise control of RNA processing by RBPs is incredibly important for cellular homeostasis, defects of which lead to numerous accounts of human genetic diseases in many tissues. With the emergence of genome-wide methods for detecting direct binding of RBPs on target RNAs, coupled with technologies to measure changes in various aspects of RNA processing, global rules and insights for individual RBPs have been revealed. However, few studies have combined regulatory changes for more than one RBP to better understand their combinatorial effects on RNA targets. Additionally, despite the importance of RBPs, we still do not have the complete repertoire of which proteins bind to RNA, and which of these bind simultaneously with other RBPs to constitute the "RBP-RNA interactome". Here, I conduct genome-wide RNA processing analyses in mammalian cells, integrating regulation and binding information for multiple RBPs, including disease-related RBPs, the highly abundant heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, and many previously uncharacterized RBPs. This battery of computational and experimental assays provides insight into the unique roles of hundreds of individual RBPs, as well as the extent of coordinated regulation between RBPs. I also describe a systematic approach to identifying the proteins that interact with RNA simultaneously with hnRNP proteins. Not limited to only mRNA-bound proteins, my strategy identifies thousands of hnRNP protein interactors, including putative novel proteins that interact with mRNA and pre-mRNA, and have biochemical and statistical attributes of known RBPs. My findings expand the repertoire of RNA-interacting RBPs and provide a resource for the study of human simultaneous RBP-RBP interactomes. This comprehensive analysis of RBPs investigates their specific roles in the regulation of RNA processing yielding interesting findings for the RNA biology field and insights into how misregulation can impact human disease.
Author: Stephanie C. Huelga
Publisher:
ISBN: 9781303990236
Category :
Languages : en
Pages : 221
Get Book Here
Book Description
Nascent transcripts produced by RNA polymerase II in eukaryotic cells are subject to extensive processing prior to the generation of a functional messenger RNA (mRNA). These RNAs are generally found coated with RNA binding proteins (RBPs), which act in concert to regulate RNA processing events, including splicing, polyadenylation, and RNA stability. The aggregate effect of various RBPs on a given RNA transcript eventually dictates its fate, a phenomenon referred to as the RNP code. The precise control of RNA processing by RBPs is incredibly important for cellular homeostasis, defects of which lead to numerous accounts of human genetic diseases in many tissues. With the emergence of genome-wide methods for detecting direct binding of RBPs on target RNAs, coupled with technologies to measure changes in various aspects of RNA processing, global rules and insights for individual RBPs have been revealed. However, few studies have combined regulatory changes for more than one RBP to better understand their combinatorial effects on RNA targets. Additionally, despite the importance of RBPs, we still do not have the complete repertoire of which proteins bind to RNA, and which of these bind simultaneously with other RBPs to constitute the "RBP-RNA interactome". Here, I conduct genome-wide RNA processing analyses in mammalian cells, integrating regulation and binding information for multiple RBPs, including disease-related RBPs, the highly abundant heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, and many previously uncharacterized RBPs. This battery of computational and experimental assays provides insight into the unique roles of hundreds of individual RBPs, as well as the extent of coordinated regulation between RBPs. I also describe a systematic approach to identifying the proteins that interact with RNA simultaneously with hnRNP proteins. Not limited to only mRNA-bound proteins, my strategy identifies thousands of hnRNP protein interactors, including putative novel proteins that interact with mRNA and pre-mRNA, and have biochemical and statistical attributes of known RBPs. My findings expand the repertoire of RNA-interacting RBPs and provide a resource for the study of human simultaneous RBP-RBP interactomes. This comprehensive analysis of RBPs investigates their specific roles in the regulation of RNA processing yielding interesting findings for the RNA biology field and insights into how misregulation can impact human disease.
Author: Soma Dash
Publisher:
ISBN: 9780438423435
Category :
Languages : en
Pages : 156
Get Book Here
Book Description
Eye development in vertebrates is initiated in late gastrulation and involves coordinated morphogenesis between the optic vesicle and the non-neural surface ectoderm resulting in the formation of the neural retina and the lens, respectively. While transcription and signaling events required for eye development are well understood, post-transcriptional control of gene expression, especially mediated by RNA-binding proteins (RBPs) is less clear. This represents a significant knowledge-gap as RBPs are important regulatory molecules in the cell that can control the fate of their target mRNAs by interacting with them throughout the mRNA life-cycle and mediating their processing, intra-cellular transport and localization, stability, translation into protein, and ultimately, their degradation. This is also a significant knowledge gap because there are similar number of RBPs encoded by the human genome as there are transcription factors, but the former class of proteins are not as well understood in the context of organogenesis and birth defects as compared to the latter. ☐ While high-throughput sequencing has identified several RBPs to be expressed in the eye, the functional significance in eye development for the vast majority of these factors is yet to be determined. Recently, the Lachke laboratory has identified two conserved RBPs required for eye development, Tdrd7 and Celf1, whose deficiency in the lens results in cataract in vertebrates. To further investigate the importance of RBP-mediated post-transcriptional gene expression control in eye development, I applied a systems-based bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to identify two new RBPs, Rbm24 and Caprin2, which are enriched during early mouse lens development, but whose molecular function in eye development had thus far not been determined. In this research dissertation, I have characterized the function of both Rbm24 and Caprin2 using constitutive and conditional targeted gene deletion mouse models. Further, in collaboration with Dr. Diane Slusarski’s laboratory (University of Iowa), zebrafish rbm24a knockout (by CRISPR/Cas9) and knockdown (by morpholino) mutants were generated and characterized. Together, these findings have led to a comprehensive understanding of the function of these RBPs in vertebrate eye development. ☐ Rbm24-targeted deletion in mouse and rbm24a-CRISPR/Cas9-targeted knockout or morpholino-knockdown in zebrafish causes the developmental defects microphthalmia (small eye) or anophthalmia (no eye). Rbm24 deficiency leads to apoptotic defects in the mouse ocular tissue as well as downregulation of eye development markers such as Sox2, Lhx2, Jag1, E-cadherin and g-Crystallins. Further, similar to the observations in the mouse, sox2 expression is also found to be reduced in rbm24a-morphant zebrafish, indicating the conservation of the Rbm24-Sox2 regulatory module in vertebrate eye development. About 20% of human anophthalmia cases are linked to SOX2 mutations alone. Therefore, I focused on investigating the post-transcriptional molecular mechanism of Rbm24-mediated Sox2 regulation. Sox2 is an intronless gene whose encoded mRNA contains AU-rich regions (ARE) in its 3’UTR. Interestingly, Rbm24 is known to bind to ARE sites in target mRNA. Therefore, to test if Rbm24 directly binds to Sox2 mRNA in vitro and in vivo, I performed RNA-Electrophoretic Mobility Shift assay (EMSA) and RNA-Immunoprecipitation (RIP), respectively. RNA-EMSA showed that Rbm24 protein directly binds to a 20 bp oligomer based on the mouse Sox2 mRNA sequence, and that an intact ARE is necessary for this protein-RNA binding. In turn, RIP assay on E14.5 wildtype mouse ocular tissue suggests that Rbm24 directly binds to Sox2 mRNA in vivo in eye development. To understand the biological significance of this direct Rbm24 protein-Sox2 mRNA molecular interaction, I performed an RNA-decay assay in NIH3T3 cells by co-transfected them with an Rbm24-overexpression vector and a Renilla luciferase reporter vector. In this assay, the Renilla luciferase gene ORF (open reading frame) is fused with the mouse Sox2 mRNA 3’UTR, which contains the three intact ARE sites, and reporter transcripts were quantified after Actinomycin-D treatment to transfected cells. This analysis demonstrates that in conditions of Rbm24 over-expression, the intact Sox2 3’UTR can render increased stability to the reporter transcript. Thus, Rbm24 positively controls Sox2 expression by binding to ARE sites in its 3’UTR and increasing its mRNA stability. Further, mutation analysis in the RNA-decay assay extends the in vitro observation that the binding of Rbm24 to the Sox2 mRNA 3’UTR depends on ARE by providing in vivo evidence that the presence of the ARE sites is necessary for the stability effect rendered by the Sox2 mRNA 3’UTR upon Rbm24 overexpression. Further, because Sox2 is one of the original four Yamanaka pluripotency/cellular reprogramming factor (along with Oct4, Klf4 and c-Myc), I investigated the impact of Rbm24 on the expression of other reprogramming factors such as Oct4, Klf4, c-Myc as well as, Nanog, another established pluripotency factor. I find that over-expression of Rbm24 in several different cell lines such as NIH3T3 (mouse embryo fibroblast cell line), 21EM15 (mouse lens epithelial cell line) and C2C12 (mouse myoblast cell line) results in the up-regulation of Sox2, Oct4 and Klf4. Further, in Rbm24-overexpressed C2C12 cells, Nanog and c-Myc are also upregulated. These data highlight that Rbm24 mediates post-transcriptional control of key transcription and pluripotency factors in vertebrate development. ☐ To gain insight into the function of the other newly identified RBP, Caprin2, in lens biology, I first performed expression analysis of Caprin2 in mouse lens development using in situ hybridization, western blotting and immunostaining. These experiments validate the iSyTE prediction that Caprin2 mRNA and protein are highly expressed and enriched in mouse embryonic and postnatal lens. I generated lens-specific Caprin2 conditional knockout (cKO) mouse mutants using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mice, wherein Caprin2 is expected to be deleted in the lens starting from E9.5 due to Cre-mediated re-arrangement of the Caprin2 alleles, revealed two distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2cKO/cKO mutants have an abnormally compact “lens nucleus”, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Further, at a reduced penetrance (8%), I find that Caprin2cKO/cKO mutants exhibit an ocular defect wherein the lens and the cornea remain attached by a persistent stalk, resembling the human developmental defect termed Peters anomaly. These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development. ☐ Together the findings in this dissertation have demonstrated that conserved RBPs such as Rbm24 and Caprin2 have evolved distinct functions in vertebrate eye development and their deficiency leads to microphthalmia and anophthalmia, and lens defects and Peters anomaly, respectively, thus impacting the study of ocular defects in humans.
Author: Kiyoshi Nagai
Publisher: Oxford University Press, USA
ISBN:
Category : Medical
Languages : en
Pages : 302
Get Book Here
Book Description
The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.
Author: A. S. N. Reddy
Publisher: Springer Science & Business Media
ISBN: 3540767762
Category : Science
Languages : en
Pages : 323
Get Book Here
Book Description
During the last few years, tremendous progress has been made in understanding various aspects of pre-mRNA processing. This book, with contributions from leading scientists in this area, summarizes recent advances in nuclear pre-mRNA processing in plants. It provides researchers in the field, as well as those in related areas, with an up-to-date and comprehensive, yet concise, overview of the current status and future potential of this research in understanding plant biology.
Author:
Publisher: Academic Press
ISBN: 0128171944
Category : Medical
Languages : en
Pages : 444
Get Book Here
Book Description
RNA-based Regulation in Human Health and Disease offers an in-depth exploration of RNA mediated genome regulation at different hierarchies. Beginning with multitude of canonical and non-canonical RNA populations, especially noncoding RNA in human physiology and evolution, further sections examine the various classes of RNAs (from small to large noncoding and extracellular RNAs), functional categories of RNA regulation (RNA-binding proteins, alternative splicing, RNA editing, antisense transcripts and RNA G-quadruplexes), dynamic aspects of RNA regulation modulating physiological homeostasis (aging), role of RNA beyond humans, tools and technologies for RNA research (wet lab and computational) and future prospects for RNA-based diagnostics and therapeutics. One of the core strengths of the book includes spectrum of disease-specific chapters from experts in the field highlighting RNA-based regulation in metabolic & neurodegenerative disorders, cancer, inflammatory disease, viral and bacterial infections. We hope the book helps researchers, students and clinicians appreciate the role of RNA-based regulation in genome regulation, aiding the development of useful biomarkers for prognosis, diagnosis, and novel RNA-based therapeutics. Comprehensive information of non-canonical RNA-based genome regulation modulating human health and disease Defines RNA classes with special emphasis on unexplored world of noncoding RNA at different hierarchies Disease specific role of RNA - causal, prognostic, diagnostic and therapeutic Features contributions from leading experts in the field
Author:
Publisher:
ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
Get Book Here
Book Description
Author:
Publisher:
ISBN: 9781071608340
Category : Gene expression
Languages : en
Pages :
Get Book Here
Book Description
Author: Gene W. Yeo
Publisher: Springer
ISBN: 1493912216
Category : Science
Languages : en
Pages : 474
Get Book Here
Book Description
After transcription in the nucleus, RNA binding proteins (RBPs) recognize cis-regulatory RNA elements within pre-mRNA sequence to form mRNA-protein (mRNP) complexes. Similarly to DNA binding proteins such as transcription factors that regulate gene expression by binding to DNA elements in the promoters of genes, RBPs regulate the fate of target RNAs by interacting with specific sequences or RNA secondary structural features within the transcribed RNA molecule. The set of functional RNA elements recognized by RBPs within target RNAs and which control the temporal, functional and spatial dynamics of the target RNA define a putative “mRNP code”. These cis-regulatory RNA elements can be found in the 5’ and 3’ untranslated regions (UTRs), introns, and exons of all protein-coding genes. RNA elements in 5’ and 3’ UTRs are frequently involved in targeting RNA to specific cellular compartments, affecting 3’ end formation, controlling RNA stability and regulating mRNA translation. RNA elements in introns and exons are known to function as splicing enhancers or silencers during the splicing process from pre-mRNA to mature mRNA. This book provides case studies of RNA binding proteins that regulate aspects of RNA processing that are important for fundamental understanding of diseases and development. Chapters include systems-level perspectives, mechanistic insights into RNA processing and RNA Binding proteins in genetic variation, development and disease. The content focuses on systems biology and genomics of RNA Binding proteins and their relation to human diseases.
Author: Robert E. Rhoads
Publisher: Humana
ISBN: 9781493936236
Category : Medical
Languages : en
Pages : 0
Get Book Here
Book Description
This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.
Author: Raymond F. Gesteland
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 744
Get Book Here
Book Description
