Mirna Maturation
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Mirna Maturation
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Author : Christoph Arenz
language : en
Publisher:
Release Date : 2014
Mirna Maturation written by Christoph Arenz and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2014 with Biomedicine categories.
In miRNA Maturation: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study miRNA maturation. These included established methods such as fluorescent and non-fluorescent methods for homogenous assays of Dicer-mediated miRNA maturation or an in vivo assay for Drosha activity. Moreover, the volume also contains useful, but less-common methods that are hard to find elsewhere. 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, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, miRNA Maturation: Methods and Protocols seeks to widen the view on miRNA as biological mediator and potential drug target.
Processing Activity Of The Mirna Maturation Endonucleases Drosha And Dicer Toward Let 7 Substrates
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Author : Gunjan Dadhwal
language : en
Publisher:
Release Date : 2022
Processing Activity Of The Mirna Maturation Endonucleases Drosha And Dicer Toward Let 7 Substrates written by Gunjan Dadhwal and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2022 with categories.
The let-7 family of microRNAs (miRNAs) comprises of thirteen members that play critical roles in many biological processes, including cell differentiation and development. More specifically, they function as tumor suppressors by targeting several oncogenes. Deregulation in let-7 miRNA levels has been associated with various human diseases, including cancers and neurodegenerative disorders. It is well established that Drosha and Dicer are the two key enzymes of the miRNA maturation pathway, and that faulty processing by these endoribonucleases could affect gene silencing. Thus, it is crucial to better understand how Drosha and Dicer respectively process the primary miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs) to yield mature miRNAs, and how these enzymes are regulated. In the last decade of miRNA research, several investigations have identified RNA structural features and RNA-binding proteins that regulate the miRNA maturation pathway, adding another layer of regulation in this pathway. However, the molecular detail of this regulation requires further investigations. The main goal of this thesis is to investigate the in vitro processing activity of Drosha and Dicer toward their let-7 substrates, focusing on how diverse sequence and structural features affect their activities. First, SHAPE structural probing followed by detailed thermodynamic and kinetic investigations for all thirteen pre-miRNAs of the let-7 family were performed with in vitro purified Dicer. Surprisingly, this study revealed that despite structural differences in the pre-let-7 members, Dicer does not discriminate between these substrates, including pre-miRNAs with a 1 nt and a 2-nt overhang at their 3'-end. Additional binding and cleavage investigations of pre let-7 substrates carrying 3'-end modifications (mono- and oligo-uridylation, mono- and oligo-adenylation) were performed to clarify how these modifications affect Dicer binding and cleavage activities. Together, this work highlights the remarkable substrate promiscuity of Dicer toward diverse pre-miRNAs of the let-7 family. Second, the enzymatic mechanism of pre-let-7 cleavage by Dicer was examined using pre-let-7a-1 as a model substrate. The results from the steady-state, pre-steady state and pulse-chase kinetics are consistent with the prevailing view, supported by recent cryo-EM structures, that the conformational change(s) of an enzyme-substrate complex into a catalytically productive conformation are important for cleavage activity. Third, the sequence and structural determinants of pri-let-7 processing by the Microprocessor (MP) complex composed of Drosha and its obligatory partner DGCR8 were investigated. Based on cleavage studies of several pri-let-7 substrates with an in vitro reconstituted MP complex, it was found that cleavage of pri-let-7g yields multiple products. Using pri-let-7g variants, it was revealed that a conserved structural element of pri-let-7g promotes unproductive cleavage, possibly as a result of the MP cleaving its substrate in the reverse orientation. This study provides the framework for future investigations in studying pri-let-7g processing by Drosha and possibly identifying novel mechanisms of regulation. Overall, our findings provide insights on how the structural features of pri-miRNAs and pre-miRNAs of the let-7 family modulate processing by Drosha and Dicer and pave the way for future studies aimed at examining the role of protein factors in regulating the maturation of let-7 miRNAs.
Micrornas In Development
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Author : Eran Hornstein
language : en
Publisher: Academic Press
Release Date : 2012-03-02
Micrornas In Development written by Eran Hornstein and has been published by Academic Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-03-02 with Medical categories.
This new volume in the Current topics in Developmental Biology series concentrates on MicroRNAs in Development. It includes chapters on such topics as miRNA networks in neuronal development, let-7 in development, and Hox networks and miRNA. With an international team of authors, this volume is a must-have addition for researchers and students alike. Concentrates on microRNAs in development Includes chapters on such topics as miRNA networks in neuronal development, let-7 in development, and Hox networks and miRNA With an international team of authors, this volume is a must-have addition for researchers and students alike
Primary Microrna Processing In Humans Biology Biotechnology And Disease
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Author : Jen Quick-Cleveland
language : en
Publisher:
Release Date : 2017
Primary Microrna Processing In Humans Biology Biotechnology And Disease written by Jen Quick-Cleveland and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017 with categories.
MicroRNAs (miRNAs) are a class of non-coding RNAs that tune gene expression by negatively regulating at least 60% of protein-coding genes. In animals, they are required for development and cell physiology. Due to their role in controlling gene expression, dysregulation of miRNA biogenesis can cause genetic disorders, immunity deficits, neurological problems, and cancers. miRNA genes are transcribed into primary miRNA (pri-miRNAs), which undergo a multi-step maturation process. The first step occurs in the nucleus, where the pri-miRNA is cleaved by the Microprocessor Complex (MC). The MC contains the ribonuclease Drosha and an RNA-binding hemoprotein DGCR8. The MC specifically recognizes the characteristic pri-miRNA hairpin to initiate miRNA maturation. This thesis centers on the nuclear step of miRNA biogenesis. My research has two main areas of focus; 1) dissecting the molecular and structural determinants that govern pri-miRNA processing, and 2) investigating the regulation of pri-miRNA processing in normal physiology and diseases. The MC identifies pri-miRNA substrates from a myriad of other RNAs. DGCR8 plays an important role in pri-miRNA recognition, but the mechanism was unknown. DGRC8 contains two double-stranded RNA-binding domains (dsRBDs), but these domains do not provide specificity. It has been shown that "junctions" between single-stranded and double-stranded regions in pri-miRNAs are important features that define MC substrates. However, the protein moiety that recognizes pri-miRNA junctions had not been identified. We discovered that DGCR8 contains an RNA-binding heme domain (Rhed) that directly and specifically binds pri-miRNA junctions. Further, we showed that the Rhed and its RNA-binding surface are required for efficient pri-miRNA processing. Previous studies of our lab showed that the pri-miRNA processing activity of DGCR8 specifically requires heme in its Fe(III) redox state. However, it is unknown how much Fe(III) heme is available in cells and how much is required to support pri-miRNA processing. During our investigation of the Rhed-pri-miRNA interface, we performed mutagenesis on DGCR8 that led to identification of single mutations with reduced heme affinity but nearly full processing activity in cells. This meant that the Fe(III) heme affinities of these mutants are sufficient for them to acquire heme and to process pri-miRNAs. In contrast, all heme-binding-deficient mutants of DGCR8 we previously characterized are inactive in cells and have lower affinities for Fe(III) heme. We discovered that the Fe(III) heme affinity threshold for activating DGCR8 is shifted by overall heme availability changes in cells. These results indicate the presence of an available ferric heme pool that distinctly determines pri-miRNA processing efficiency in cells. Our study suggests cellular redox state and currently unknown Fe(III) heme-specific transporter proteins may be important for regulating miRNA maturation. Secondary structure is a defining characteristic of pri-miRNA substrates. Substantial effort has been made to identify the important features of processing-competent pri-miRNA hairpins. Little attention is paid to structures outside the canonical hairpin. We identified a helix (f-helix) flanking the pri-miR-30a hairpin. Using an in vitro processing assay, we found that disrupting the pairing in this structure caused processing defects that could be rescued by repairing the helix. Although f-helices are not found in all pri-miRNAs, our finding has important implications on how to improve DNA vector-based RNA interference technology. The second generation short-hairpin RNAs (shRNAmir) are designed to mimic pri-miRNA and thus produce small RNAs through the natural miRNA maturation pathway. Their structure is most often based on pri-miR-30a. Indeed, we showed that the f-helix was functionally important for shRNAmir processing and knockdown efficiency. This work indicates that structural elements surrounding the pri-miRNA hairpin may serve as regulatory elements for miRNA maturation. miRNA expression is globally repressed in many tumors, and this dysregulation can drive tumorigenesis. High numbers of somatic mutations are a hallmark of many tumors, but distinguishing between disease-driving and spurious mutations remains a challenge. We analyzed seven missense DGCR8 mutations and found that four are severely defective. We determined that the E518K mutation identified in about 3% of Wilms Tumors, most likely disrupts the folding of DGCR8. The F448L mutation found in colon cancers rendered DGCR8 unable to bind Fe (III) heme and the Drosha protein. This result suggests that heme-binding to DGCR8 may be required for strong association with Drosha. Biochemical characterization of K289E and G336E show they have reduced affinities both pri-miRNA and heme. Altogether, these results suggest that a substantial fraction of tumor-derived DGCR8 mutations results in functional deficits and thereby are likely to make important contribution to a cancer-promoting phenotype. Overall, my thesis investigates the fundamental mechanism of pri-miRNA processing, and its applications to biotechnology and diseases. My main scientific contribution is the identification and characterization of the unique RNA-binding heme domain in DGCR8. My work demonstrates the importance of heme in pri-miRNA processing, and defines an Fe(III) heme pool that regulates MC activity, thereby linking miRNA biogenesis with heme biology and cellular redox environments. My research extends our understanding of the functional/regulatory elements in pri-miRNA hairpins to include regions flanking the conserved pri-miRNA hairpin. Finally, I identify probable driver mutations for tumor formation by connecting several cancer-associated somatic point-mutants of DGCR8 to functional defects in processing.
Microrna In Development And In The Progression Of Cancer
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Author : Shree Ram Singh
language : en
Publisher: Springer Science & Business
Release Date : 2014-04-21
Microrna In Development And In The Progression Of Cancer written by Shree Ram Singh and has been published by Springer Science & Business this book supported file pdf, txt, epub, kindle and other format this book has been release on 2014-04-21 with Medical categories.
miRNAs are a class of endogenous, small non-protein coding RNA molecules (~ 22 nucleotides) which are novel post-transcriptional regulators of gene expression. Since we have hundreds of miRNAs, the major challenge is now to understand their specific biological function. In fact the experimental evidence suggests that signaling pathways could be ideal candidates for miRNA-mediated regulation. Several studies suggest that miRNAs affect the responsiveness of cells to signaling molecules such as WNT, Notch, TGF-β and EGFR. Altered expression of particular miRNAs has been implicated in the onset and development of cancer and could be used as potential biomarkers for the disease. Recently, many studies have found miRNAs have crucial regulatory roles in Cancer stem cells (CSCs) a kind of tumor initiating cells (TICs) and dormancy. Findings also suggest that DNA methylation may be important in regulating the expression of many miRNAs in several cancer initiating cells. Several miRNAs are known to either upregulated or downregulated in CSCs when compared to non-cancerous cells from the same tissues. CSCs are a small subpopulation of cells identified in a variety of tumors and involve in self-renewal, differentiation, chemoresistance and tumorigenesis. The volume will give a comprehensive account of important advancements in the area of miRNAs and cancer.
Regulation Of Let 7 Mirna Biogenesis In C Elegans
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Author : Zoya Kai
language : en
Publisher:
Release Date : 2011
Regulation Of Let 7 Mirna Biogenesis In C Elegans written by Zoya Kai and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2011 with categories.
The let-7 miRNA is a highly conserved regulatory molecule that dictates development across animal phyla, and mis-regulation of let-7 in humans results in numerous diseases. In order to unravel the complex mechanisms involved in the biogenesis of the let-7 microRNA (miRNA), my research has focused on three areas: transcriptional regulation of primary let-7 (pri-let-7) throughout development, post-transcriptional regulation of pri-let-7 by the LIN-28 protein during early developmental stages, and post-transcriptional regulation of pri-let-7 during later developmental stages by the mature let-7 miRNA and ALG-1 protein (Argonaute Like Gene 1). In the first, and primary, area of my research--transcriptional regulation of let-7--I have discovered a novel cis-element in the promoter of the let-7 gene, trans-acting factors regulating the spatio-temporal specificity of pri-let-7 transcription and a dynamically cycling transcriptional expression regulated in the molting pathway. Investigating post-transcriptional regulation of let-7 biogenesis, I collaborated with Dr. Priscilla Van Wynsberge and we found that LIN-28, a highly conserved RNA binding protein, co-transcriptionally associates with pri-let-7 to negatively regulate pri-let-7 biogenesis. Working with Dr. Dimitrios G. Zisoulis, we discovered that the let-7 miRNA guided miRISC (miRNA Induced Silencing Complex) with the ALG-1 protein, directly binds and positively regulates maturation of pri-let-7 transcripts in a novel auto-regulatory feedback loop that is conserved across species. This is the first report of the miRISC complex targeting a primary miRNA transcript. This auto-regulatory model of miRNA maturation provides a novel role for ALG-1 as well as let-7 and introduces a new mechanism of miRNA regulation.
Drug Target Mirna
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Author : Marco F. Schmidt
language : en
Publisher: Humana Press
Release Date : 2018-07-06
Drug Target Mirna written by Marco F. Schmidt and has been published by Humana Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2018-07-06 with Medical categories.
This volume provides a concise and technical discussion of recently developed approaches to overcome challenges, such as pharmacodynamics and pharmacokinetics difficulties, in miRNA drug discovery. These strategies cover anti-sense agents targeting miRNA that are applied in advanced formulations or are chemically optimized to increase delivery; small molecule miRNA modulators to overcome anti-sense agents’ limitations; general enhancers of miRNA maturation; and Argonaute 2 protein and its pharmacokinetic parameters. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Drug Target miRNA: Methods and Protocols is a valuable resource for anyone interested in the ever-evolving field of miRNA drug discovery.
Micrornas In Development And Cancer
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Author : Frank J. Slack
language : en
Publisher: World Scientific
Release Date : 2011
Micrornas In Development And Cancer written by Frank J. Slack and has been published by World Scientific this book supported file pdf, txt, epub, kindle and other format this book has been release on 2011 with Science categories.
MicroRNAs have recently emerged as key regulators of gene expression during development and are frequently misexpressed in human disease states, in particular cancer. These 22-nucleotide-long transcripts act to promote or repress cell proliferation, migration and apoptosis during development, all of which are processes that go awry in cancer. Thus, microRNAs have the ability to behave like oncogenes or tumor suppressors. In addition, their small size and molecular properties make them amenable as targets and therapeutics in cancer treatment. This book goes into detail on how microRNAs represent a paradigm shift in thinking about gene regulation during development and disease, and provide the oncologist with a potentially powerful new battery of agents to diagnose and treat cancer.
Micrornas In Plant Development And Stress Responses
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Author : Ramanjulu Sunkar
language : en
Publisher: Springer Science & Business Media
Release Date : 2012-02-22
Micrornas In Plant Development And Stress Responses written by Ramanjulu Sunkar and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2012-02-22 with Science categories.
Precise regulation of gene expression in both time and space is vital to plant growth, development and adaptation to biotic and abiotic stress conditions. This is achieved by multiple mechanisms, with perhaps the most important control being exerted at the level of transcription. However, with the recent discovery of microRNAs another ubiquitous mode of gene regulation that occurs at the post-transcriptional level has been identified. MicroRNAs can silence gene expression by targeting complementary or partially complementary mRNAs for degradation or translational inhibition. Recent studies have revealed that microRNAs play fundamental roles in plant growth and development, as well as in adaptation to biotic and abiotic stresses. This book highlights the roles of individual miRNAs that control and regulate diverse aspects of plant processes.
Exploring Rna Flexibility Using Molecular Dynamics
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Author : Katheryn Penrod
language : en
Publisher:
Release Date : 2017
Exploring Rna Flexibility Using Molecular Dynamics written by Katheryn Penrod and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2017 with categories.
Non-coding microRNAs (miRNAs) have been identified as powerful regulators of gene expression. Approximately 22 nucleotides in length, these RNAs are found in most eukaryotes, including humans. Through a process called RNA interference (RNAi), mature miRNA binds target mRNA molecules to accomplish gene silencing. Recently, miRNA has emerged as a potential therapeutic target for various disease states that have been linked to changes in miRNA expression.In humans, miRNA is produced in the form of a stem-loop pri-miRNA structure containing approximately 100 nucleotides. Through a series of binding and cleavage events collectively referred to as the miRNA maturation pathway, this primary miRNA (pri-miRNA) is converted to the single-stranded mature miRNA that participates in RNAi. The underlying molecular processes of the miRNA maturation pathway are not completely understood. Structural characterization of the proteins and nucleic acids along this pathway will contribute to a deeper understanding of miRNA that would be particularly beneficial in the pharmaceutical industry. Five proteins along the miRNA maturation pathway possess at least one double-stranded RNA binding domain (dsRBD) responsible for facilitating protein-RNA interactions. These evolutionarily-conserved domains form non-bonded interactions with the phosphodiester backbone and 2-hydroxyl groups within the minor groove of dsRNA. Although the precise mechanism remains unclear, dsRBDs generally recognize their substrates in a shape-dependent and non sequence-specific manner.The TAR RNA binding protein (TRBP) is responsible for binding precursor miRNA (pre-miRNA) and presenting it to Dicer for cleavage. In a previous study, binding by TRBP was demonstrated to exclude sites of helical imperfections. The ubiquity of such imperfections in miRNA suggests that dsRBDs can sense these structural features in order ensure the proper orientation of their substrates for cleavage. The overarching aim of this work was to characterize the binding events along the miRNA maturation pathway with respect to RNA flexibility. Initially, we use circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular dynamics (MD) simulations to investigate a simplified system containing TRBP-dsRBD2 and a perfect WC duplex of 20 GC base pairs. Strong protein-RNA contacts were observed in expected regions of the complex, supporting the previous notion that TRBP preferentially binds to perfect WC duplexes. Although cocrystal structures suggest that binding by TRBP and Xlrbpa-2 induces strong bends in coaxially-stacked GC10-mers, no large-scale conformational changes were detected in the TRBP / GC20 complex. We conclude that the bending observed in the cocrystal structures is most likely a result of the artificial double-stranded break between the oligomers. Having established a suitable method for performing and analyzing MD simulations of a simple dsRBD / dsRNA complex, we designed a more realistic system. We selected pri-mir-16-1 for this study based on the SHAPE-constrained MC-Pipeline structures previously determined by our laboratory. The three-dimensional structure model of pri-mir-16-1 reveals two deformable hot spots. The deformable region adjacent to the Drosha cut site was observed in other pri-miRNAs and was demonstrated to influence processing efficiency. A second region was identified for pri-mir-16-1 in the vicinity of its A37 / A76 mismatch and nearby U79 bulge. It has been suggested that structural distortions of this type promote DGCR8 binding by allowing the formation of strong axial bends. Toward complete characterization of this binding event, unbound pri-mir-16-1 was simulated using the same protocol as above. Preliminary results indicate that the procedures for trajectory analysis require modification to accommodate structural imperfections. Further investigation of this system will illuminate the effect of structural imperfections on the conformational flexibility of dsRNA and provide a reliable means of investigating similar interactions along the miRNA maturation pathway.