Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae: Cloning, expression, purification, and activity assays Journal Article
| Authors: | Greimann, J. C.; Lima, C. D. |
| Article Title: | Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae: Cloning, expression, purification, and activity assays |
| Abstract: | Eukaryotic RNA exosomes participate in 3′ to 5′-processing and degradation of RNA in the nucleus and cytoplasm. RNA exosomes are multisubunit complexes composed of at least nine distinct proteins that form the exosome core. Although the eukaryotic exosome core shares structural and sequence similarity to phosphorolytic archaeal exosomes and bacterial PNPase, the eukaryotic exosome core has diverged from its archaeal and bacterial cousins and appears devoid of phosphorolytic activity. In yeast, the processive hydrolytic 3′ to 5′-exoribonuclease Rrp44 associates with exosomes in the nucleus and cytoplasm. Although human Rrp44 appears homologous to yeast Rrp44, it has not yet been shown to associate with human exosomes. In the nucleus, eukaryotic exosomes interact with Rrp6, a distributive hydrolytic 3′ to 5′-exoribonuclease. To facilitate analysis of eukaryotic RNA exosomes, we will describe procedures used to clone, express, purify, and reconstitute the nine-subunit human exosome and nine-, ten-, and eleven-subunit yeast exosomes. We will also discuss procedures to assess exoribonuclease activity for reconstituted exosomes. © 2008 Elsevier Inc. All rights reserved. |
| Keywords: | protein expression; protein phosphorylation; genetics; review; nonhuman; methodology; polymerase chain reaction; protein domain; animal; metabolism; animals; gene expression; protein degradation; bacteria (microorganisms); rna; molecular cloning; cloning, molecular; chemistry; protein processing; hybrid protein; protein purification; saccharomyces cerevisiae; eukaryota; substrate specificity; recombinant protein; cell nucleus; saccharomyces cerevisiae proteins; protein subunit; saccharomyces cerevisiae protein; eukaryotic cell; sequence homology; exonuclease; enzyme specificity; isolation and purification; cell interaction; archaea; fungal protein; complementary dna; exonucleases; exosome; exosomes; hydrolysis kinetics |
| Journal Title: | Methods in Enzymology |
| Volume: | 448 |
| ISSN: | 0076-6879 |
| Publisher: | Academic Press |
| Date Published: | 2008-01-01 |
| Start Page: | 185 |
| End Page: | 210 |
| Language: | English |
| DOI: | 10.1016/s0076-6879(08)02610-4 |
| PUBMED: | 19111177 |
| PROVIDER: | scopus |
| PMCID: | PMC2773543 |
| DOI/URL: | |
| Notes: | Chapter 10 in "RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay" (ISBN: 978-0-12-374378-7) - Cited By (since 1996): 4 - Export Date: 17 November 2011 - CODEN: MENZA - Source: Scopus |
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