Isolation of mRNAs associated with yeast mitochondria to study mechanisms of localized translation

Chen Lesnik, Yoav Arava

Research output: Contribution to journalArticlepeer-review

Abstract

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes' association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Original languageEnglish
Article numbere51265
JournalJournal of Visualized Experiments
Issue number85
DOIs
StatePublished - 14 Mar 2014
Externally publishedYes

Keywords

  • Biochemical fractionation
  • Biochemistry
  • Issue 85
  • Localized translation
  • Microarray
  • Mitochondria
  • S. cerevisiae
  • Yeast
  • mRNA localization

ASJC Scopus subject areas

  • General Neuroscience
  • General Chemical Engineering
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology

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