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Analysis of rrna processing and translation in mammalian cells using a synthetic 18s rrna expression system

Academic Article
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Overview

authors

  • Burman, L. G.
  • Mauro, Vincent

publication date

  • 2012

journal

  • Nucleic Acids Research  Journal

abstract

  • Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈ 15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5' external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

subject areas

  • Animals
  • Cell Line
  • DNA, Ribosomal Spacer
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Pactamycin
  • Protein Biosynthesis
  • Protein Synthesis Inhibitors
  • RNA Precursors
  • RNA Processing, Post-Transcriptional
  • RNA, Ribosomal, 18S
  • Ribosome Subunits, Small, Eukaryotic
  • Sequence Deletion
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Identity

PubMed Central ID

  • PMC3439915

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/gks530

PubMed ID

  • 22718970
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Additional Document Info

start page

  • 8085

end page

  • 8098

volume

  • 40

issue

  • 16

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