Effects on mitochondrial transcription of manipulating mTERF protein levels in cultured human HEK293 cells

BMC Mol Biol. 2010 Sep 16;11:72. doi: 10.1186/1471-2199-11-72.

Abstract

Background: Based on its activities in vitro, the mammalian mitochondrial transcription termination factor mTERF has been proposed to regulate mitochondrial transcription by favouring termination at its high-affinity binding immediately downstream of the rDNA segment of mitochondrial DNA, and initiation selectively at the PH1 site of the heavy-strand promoter. This defines an rDNA transcription unit distinct from the 'global' heavy-strand transcription unit initiating at PH2. However, evidence that the relative activities of the two heavy-strand transcription units are modulated by mTERF in vivo is thus far lacking.

Results: To test this hypothesis, we engineered human HEK293-derived cells for over-expression or knockdown of mTERF, and measured the steady-state levels of transcripts belonging to different transcription units, namely tRNALeu(UUR) and ND1 mRNA for the PH2 transcription unit, and tRNAPhe plus 12S and 16S rRNA for the PH1 transcription unit. The relative levels of 16S rRNA and ND1 mRNA were the same under all conditions tested, although mTERF knockdown resulted in increased levels of transcripts of 12S rRNA. The amount of tRNAPhe relative to tRNALeu(UUR) was unaffected by mTERF over-expression, altered only slightly by mTERF knockdown, and was unchanged during recovery from ethidium bromide-induced depletion of mitochondrial RNA. mTERF overexpression or knockdown produced a substantial shift (3-5-fold) in the relative abundance of antisense transcripts either side of its high-affinity binding site.

Conclusions: mTERF protein levels materially affect the amount of readthrough transcription on the antisense strand of mtDNA, whilst the effects on sense-strand transcripts are complex, and suggest the influence of compensatory mechanisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic-Leucine Zipper Transcription Factors / genetics
  • Basic-Leucine Zipper Transcription Factors / metabolism*
  • DNA, Mitochondrial / genetics*
  • DNA, Mitochondrial / metabolism*
  • HEK293 Cells*
  • Humans
  • Mitochondrial Proteins
  • Promoter Regions, Genetic
  • RNA / genetics
  • RNA / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Mitochondrial
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism
  • RNA, Transfer, Leu / genetics
  • RNA, Transfer, Leu / metabolism
  • RNA, Transfer, Phe / genetics
  • RNA, Transfer, Phe / metabolism
  • Transcription, Genetic*

Substances

  • Basic-Leucine Zipper Transcription Factors
  • DNA, Mitochondrial
  • MTERF1 protein, human
  • Mitochondrial Proteins
  • RNA, Messenger
  • RNA, Mitochondrial
  • RNA, Ribosomal
  • RNA, Transfer, Leu
  • RNA, Transfer, Phe
  • RNA