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New Insights into the Biochemistry a...

Trotman, Jackson B.

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New Insights into the Biochemistry and Cell Biology of RNA Recapping.

Record Type:	Electronic resources : Monograph/item
Title/Author:	New Insights into the Biochemistry and Cell Biology of RNA Recapping./
Author:	Trotman, Jackson B.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
Description:	146 p.
Notes:	Source: Dissertations Abstracts International, Volume: 80-12, Section: B.
Contained By:	Dissertations Abstracts International80-12B.
Subject:	Molecular biology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13906025
ISBN:	9781392177501

New Insights into the Biochemistry and Cell Biology of RNA Recapping.
Trotman, Jackson B.

New Insights into the Biochemistry and Cell Biology of RNA Recapping. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 146 p.

Source: Dissertations Abstracts International, Volume: 80-12, Section: B.

Thesis (Ph.D.)--The Ohio State University, 2018.

This item must not be sold to any third party vendors.

Eukaryotic gene expression depends on the 5' cap structure added to all mRNAs. The biology of RNA capping is more dynamic than originally thought, with cytoplasmic recapping enabling spatial and temporal control of translation and other aspects of the RNA life cycle. Despite progress over the past decade, critical gaps remain in our understanding of the biochemistry of RNA recapping and its context within the cell, which are addressed in this dissertation.Recapping an uncapped RNA in the cytoplasm begins with the combined activities of a 5' monophosphate RNA kinase and capping enzyme (CE). Recapped RNAs are also methylated at the N7 position of the cap guanine, enabling recognition by cap-binding proteins such as the translation initiation factor eIF4E. However, it was unknown how caps synthesized in the cytoplasm acquire this critical methyl group. Here I describe the identification of the enzyme that completes the synthesis of mature caps in the cytoplasm. This enzyme, RNA guanine-7 methyltransferase (RNMT), was originally thought to be restricted to the nucleus, but I show that it also functions in the cytoplasm. Cytoplasmic RNMT activity is unexpectedly robust compared to that of nuclear RNMT, and RNMT knockdown points to RNMT being the predominant, if not only cap guanine-N7 methyltransferase in the cytoplasm. These results were obtained using an adapted cap methyltransferase activity assay, the details of which are provided. RNMT directly interacts with CE through its C-terminal catalytic domain, allowing it to associate with the multifunctional cytoplasmic capping complex. Cytoplasmic RNMT activity is additionally stimulated by dimerization with the small protein cofactor RAM. Inhibiting cytoplasmic cap methylation with a dominant-negative form of RNMT caused recapping target RNAs to destabilize, suggesting surveillance by decapping enzymes specific for unmethylated caps.The full complement of proteins required for cytoplasmic recapping is also unknown. Toward this end, I used in vivo crosslinking and bottom-up proteomics to identify novel proteins that interact with the cytoplasmic population of capping enzyme. The identified proteins perform diverse functional roles and include metabolic enzymes, nucleocytoplasmic transporters, and cytoskeletal proteins. Many of these proteins are known to bind mRNA and may underlie the observed specificity of recapping target RNAs. Western blotting confirmed the association of cytoplasmic CE with the candidate interactors FASN, XPO2, HSP90, EEF2, RUVBL1, and RUVBL2. Further investigation of HSP90 revealed that its interaction with CE causes stabilization of the levels of both nuclear and cytoplasmic CE. HSP90 stabilization may provide a link between cytoplasmic recapping and the cellular stress response, which is discussed.

ISBN: 9781392177501Subjects--Topical Terms:

517296
Molecular biology.

New Insights into the Biochemistry and Cell Biology of RNA Recapping.
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500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Schoenberg, Daniel.
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506 $a This item must not be sold to any third party vendors.
506 $a This item must not be added to any third party search indexes.
520 $a Eukaryotic gene expression depends on the 5' cap structure added to all mRNAs. The biology of RNA capping is more dynamic than originally thought, with cytoplasmic recapping enabling spatial and temporal control of translation and other aspects of the RNA life cycle. Despite progress over the past decade, critical gaps remain in our understanding of the biochemistry of RNA recapping and its context within the cell, which are addressed in this dissertation.Recapping an uncapped RNA in the cytoplasm begins with the combined activities of a 5' monophosphate RNA kinase and capping enzyme (CE). Recapped RNAs are also methylated at the N7 position of the cap guanine, enabling recognition by cap-binding proteins such as the translation initiation factor eIF4E. However, it was unknown how caps synthesized in the cytoplasm acquire this critical methyl group. Here I describe the identification of the enzyme that completes the synthesis of mature caps in the cytoplasm. This enzyme, RNA guanine-7 methyltransferase (RNMT), was originally thought to be restricted to the nucleus, but I show that it also functions in the cytoplasm. Cytoplasmic RNMT activity is unexpectedly robust compared to that of nuclear RNMT, and RNMT knockdown points to RNMT being the predominant, if not only cap guanine-N7 methyltransferase in the cytoplasm. These results were obtained using an adapted cap methyltransferase activity assay, the details of which are provided. RNMT directly interacts with CE through its C-terminal catalytic domain, allowing it to associate with the multifunctional cytoplasmic capping complex. Cytoplasmic RNMT activity is additionally stimulated by dimerization with the small protein cofactor RAM. Inhibiting cytoplasmic cap methylation with a dominant-negative form of RNMT caused recapping target RNAs to destabilize, suggesting surveillance by decapping enzymes specific for unmethylated caps.The full complement of proteins required for cytoplasmic recapping is also unknown. Toward this end, I used in vivo crosslinking and bottom-up proteomics to identify novel proteins that interact with the cytoplasmic population of capping enzyme. The identified proteins perform diverse functional roles and include metabolic enzymes, nucleocytoplasmic transporters, and cytoskeletal proteins. Many of these proteins are known to bind mRNA and may underlie the observed specificity of recapping target RNAs. Western blotting confirmed the association of cytoplasmic CE with the candidate interactors FASN, XPO2, HSP90, EEF2, RUVBL1, and RUVBL2. Further investigation of HSP90 revealed that its interaction with CE causes stabilization of the levels of both nuclear and cytoplasmic CE. HSP90 stabilization may provide a link between cytoplasmic recapping and the cellular stress response, which is discussed.
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