Biochemical Studies of the Eukaryotic RNA Exosome

Biochemical Studies of the Eukaryotic RNA Exosome PDF Author: Matthew Alan Bratkowski
Publisher:
ISBN:
Category :
Languages : en
Pages : 209

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Book Description
The RNA exosome is a multi-subunit protein complex involved in RNA maturation, surveillance, and turnover of cellular RNA. It is composed of a nine subunit core that contains an RNA binding central channel and associates with the additional subunit Rrp44. Rrp44 contains a 3' to 5' processive exoribonuclease activity and an endoribonuclease activity in its N-terminal PIN domain. The exosome is present in both the nucleus and cytoplasm. In the nucleus, the exosome associates with a second nuclease, Rrp6, which is a distributive 3' to 5' exoribonuclease. Although a wealth of studies have helped to characterize the exosome, it is still not entirely clear how the exosome core functions in concert with these two nucleases to degrade RNA. In this dissertation, I present structural and biochemical studies of both the Rrp44 and Rrp6 bound exosome. In particular, I have attempted to crystallize a sub-complex of Rrp44 bound to the exosome, and present data that suggest that structured RNA can access Rrp44 without being first channeled through the exosome. Despite this conclusion, the exosome core still appears to down-regulate both the exonuclease and endonuclease activities of Rrp44, possibly by causing the protein to assume a conformation that prohibits it from promiscuous RNA degradation. Work on the Rrp6 containing exosme was conducted in attempts of obtaining an EM reconstruction of this complex. Crystallization of an RNA and Rrp6 binding exosome associated iii protein, Rrp47, was also attempted alone and in complex with Rrp6. Overall, the results that I present here will help to further characterize the function of the exosome in RNA mediated degradation. iv.

Biochemical Studies of the Eukaryotic RNA Exosome

Biochemical Studies of the Eukaryotic RNA Exosome PDF Author: Matthew Alan Bratkowski
Publisher:
ISBN:
Category :
Languages : en
Pages : 209

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Book Description
The RNA exosome is a multi-subunit protein complex involved in RNA maturation, surveillance, and turnover of cellular RNA. It is composed of a nine subunit core that contains an RNA binding central channel and associates with the additional subunit Rrp44. Rrp44 contains a 3' to 5' processive exoribonuclease activity and an endoribonuclease activity in its N-terminal PIN domain. The exosome is present in both the nucleus and cytoplasm. In the nucleus, the exosome associates with a second nuclease, Rrp6, which is a distributive 3' to 5' exoribonuclease. Although a wealth of studies have helped to characterize the exosome, it is still not entirely clear how the exosome core functions in concert with these two nucleases to degrade RNA. In this dissertation, I present structural and biochemical studies of both the Rrp44 and Rrp6 bound exosome. In particular, I have attempted to crystallize a sub-complex of Rrp44 bound to the exosome, and present data that suggest that structured RNA can access Rrp44 without being first channeled through the exosome. Despite this conclusion, the exosome core still appears to down-regulate both the exonuclease and endonuclease activities of Rrp44, possibly by causing the protein to assume a conformation that prohibits it from promiscuous RNA degradation. Work on the Rrp6 containing exosme was conducted in attempts of obtaining an EM reconstruction of this complex. Crystallization of an RNA and Rrp6 binding exosome associated iii protein, Rrp47, was also attempted alone and in complex with Rrp6. Overall, the results that I present here will help to further characterize the function of the exosome in RNA mediated degradation. iv.

The Eukaryotic RNA Exosome

The Eukaryotic RNA Exosome PDF Author: John LaCava
Publisher: Humana
ISBN: 9781493998241
Category : Medical
Languages : en
Pages : 517

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Book Description
This volume provides a cross-section of RNA exosome research protocols, applied to a diversity of model organisms. Chapters guide readers through methods that e.g. delineate eukaryotic exosomes’ origins in prokaryotes, probe its RNA substrates, adapter complexes and macromolecular interaction of networks, and establish critical structural-function relationships. 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. Authoritative and cutting-edge, The Eukaryotic RNA Exosome: Methods and Protocols aims to ensure successful results in the further study of this vital field.

RNA Exosome

RNA Exosome PDF Author: Torben Heick Jensen
Publisher: Springer Science & Business Media
ISBN: 1441978410
Category : Medical
Languages : en
Pages : 144

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Book Description
The diversity of RNAs inside living cells is amazing. We have known of the more “classic” RNA species: mRNA, tRNA, rRNA, snRNA and snoRNA for some time now, but in a steady stream new types of molecules are being described as it is becoming clear that most of the genomic information of cells ends up in RNA. To deal with the enormous load of resulting RNA processing and degradation reactions, cells need adequate and efficient molecular machines. The RNA exosome is arising as a major facilitator to this effect. Structural and functional data gathered over the last decade have illustrated the biochemical importance of this multimeric complex and its many co-factors, revealing its enormous regulatory power. By gathering some of the most prominent researchers in the exosome field, it is the aim of this volume to introduce this fascinating protein complex as well as to give a timely and rich account of its many functions. The exosome was discovered more than a decade ago by Phil Mitchell and David Tollervey by its ability to trim the 3’end of yeast, S. cerevisiae, 5. 8S rRNA. In a historic account they laid out the events surrounding this identification and the subsequent birth of the research field. In the chapter by Kurt Januszyk and Christopher Lima the structural organization of eukaryotic exosomes and their evolutionary counterparts in bacteria and archaea are discussed in large part through presentation of structures.

Structure and Biochemistry of the Eukaryotic RNA Exosome

Structure and Biochemistry of the Eukaryotic RNA Exosome PDF Author: Jaclyn Caye Greimann
Publisher:
ISBN:
Category :
Languages : en
Pages : 210

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Eukaryotic RNases and Their Partners in RNA Degradation and Biogenesis

Eukaryotic RNases and Their Partners in RNA Degradation and Biogenesis PDF Author: Guillaume Chanfreau
Publisher: Academic Press
ISBN: 0124047408
Category : Medical
Languages : en
Pages : 278

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Book Description
This special issue of The Enzymes is targeted towards researchers in biochemistry, molecular and cell biology, pharmacology, and cancer. This volume discusses Eukaryotic RNases and their partners in RNA degradation and biogenesis. Contributions from leading authorities Informs and updates on all the latest developments in the field

Eukaryotic RNases and Their Partners in RNA Degradation and Biogenesis

Eukaryotic RNases and Their Partners in RNA Degradation and Biogenesis PDF Author:
Publisher: Academic Press
ISBN: 0124047459
Category : Science
Languages : en
Pages : 278

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Book Description
This special issue of The Enzymes is targeted towards researchers in biochemistry, molecular and cell biology, pharmacology, and cancer. This volume discusses Eukaryotic RNases and their partners in RNA degradation and biogenesis. Contributions from leading authorities Informs and updates on all the latest developments in the field

RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay

RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay PDF Author: Lynne E. Maquat
Publisher: Academic Press
ISBN: 9780080922072
Category : Science
Languages : en
Pages : 656

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Book Description
Specific complexes of protein and RNA carry out many essential biological functions, including RNA processing, RNA turnover, 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 to identify mRNA processing, transport, localization and turnover, which are central to the process of gene expression Offers step-by-step lab instructions, including necessary equipment and reagents

Advances in Immunology

Advances in Immunology PDF Author: Frederick W. Alt
Publisher: Academic Press
ISBN: 012802433X
Category : Medical
Languages : en
Pages : 332

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Book Description
Advances in Immunology, a long-established and highly respected publication, presents current developments as well as comprehensive reviews in immunology. Articles address the wide range of topics that comprise immunology, including molecular and cellular activation mechanisms, phylogeny and molecular evolution, and clinical modalities. Edited and authored by the foremost scientists in the field, each volume provides up-to-date information and directions for the future. Contributions from leading authorities Informs and updates on all the latest developments in the field

Synergy Between the Exoribonucleases Rrp6p and Rrp44p in the Nuclear Exosome Complex

Synergy Between the Exoribonucleases Rrp6p and Rrp44p in the Nuclear Exosome Complex PDF Author: Armend Axhemi
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 174

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Book Description
The 11-component nuclear exosome (Exo11) plays an essential role in eukaryotic RNA metabolism by degrading or processing nearly all classes of RNA in a 3' to 5' direction. Exo11 contains two exonucleases, Rrp6p and Rrp44p, which are located at opposite ends of a nine-component inactive core (Exo9). To perform their biological roles, the nucleases target a large number of substrates, but they do not indiscriminately degrade all RNAs. How they accomplish this selectivity was not understood. It was also unclear how Exo9 impacts the activities of nucleases, and how it integrates their activities in the 11-component complex. We measured RNA binding and degradation kinetics of exosome components in vitro at single nucleotide resolution across a diverse array of substrates. We find that Rrp6p is inherently selective. RNA length and the four 3'-terminal nucleotides contribute most to substrate selectivity of Rrp6p and enable it to discriminate between different RNAs by several orders of magnitude. The most pronounced discrimination is seen against RNAs ending with CCA-3' which correspond to 3'-termini of uncharged tRNAs. Rrp6p is processive for longer substrates but becomes distributive for shorter ones. The data also show that in contrast to many other enzymes that use substrate selectivity to preferentially interact with specific RNAs, Rrp6p uses its selectivity to discriminate against specific RNAs. Rrp44p degrades RNA in two phases. Phase one is rate limiting while phase two is fast and processive. Upstream RNA length, sequence and the four 3'-terminal nucleotides contribute to substrate selectivity of Rrp44p. These features enable Rrp44p to discriminate at binding and forward rate constants for both phases of degradation. Exo9 diminishes the activity of Rrp6p for all substrates while its impact on Rrp44p is substrate dependent. Substrate selectivity of both nucleases is left intact in the 10-component complexes. The 11-component complex (Exo11) integrates optimal features of Rrp6p and Rrp44p to create synergy. The affinity of Exo11 for certain RNAs is roughly two orders of magnitude greater than that of its constituent components for same substrates. Our findings indicate that Exo9 plays a critical role in integrating and coordinating activities to create synergy between the two exonucleases.

The Biology of mRNA: Structure and Function

The Biology of mRNA: Structure and Function PDF Author: Marlene Oeffinger
Publisher: Springer Nature
ISBN: 3030314340
Category : Science
Languages : en
Pages : 312

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Book Description
The book provides an overview on the different aspects of gene regulation from an mRNA centric viewpoint, including how mRNA is assembled and self-assembles in a complex consisting of RNA and proteins, and how its ability to be translated at the right time and space depends on many processes acting on the mRNAs, leading to a properly folded complex. This book shows how new technologies have led to a better understanding of these processes and their connected diseases.The book is written for scientists in fundamental and applied biomedical research working on different aspects of gene regulation. It is also targeted to an audience that is not implicated in these fields directly, but wants to gain a better understanding of mRNA biology.