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Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA

O Kessler1, L A Chasin

  • 1Department of Biological Sciences, Columbia University, New York, New York 10027, USA.

Molecular and Cellular Biology
|August 1, 1996
PubMed

Insights

Nonsense mutations in the adenine phosphoribosyltransferase (aprt) gene reduce mRNA levels in Chinese hamster ovary cells. Translation appears to facilitate mRNA export from the nucleus, though the exact mechanism requires further study.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Nonsense mutations can lead to reduced mRNA levels through various mechanisms.
  • The adenine phosphoribosyltransferase (aprt) gene is crucial for DNA repair and purine metabolism.
  • Understanding mRNA decay pathways is vital for comprehending gene regulation.

Purpose of the Study:

  • To investigate the impact of nonsense codons in the aprt gene on mRNA stability and expression in Chinese hamster ovary (CHO) cells.
  • To elucidate the role of translation and nuclear export in nonsense-mediated mRNA decay (NMD).

Main Methods:

  • Analysis of CHO cell mutants with nonsense codons in different exons of the aprt gene.
  • Measurement of steady-state and nuclear aprt mRNA levels.
  • Assessment of mRNA decay rates using actinomycin D and tetracycline-induced transcription inhibition.
  • Investigation of mRNA association with polysomes.
  • Quantitation of intron-containing transcripts.

Main Results:

  • Mutations in exons 1, 2, and 4 of the aprt gene caused a 5- to 10-fold reduction in steady-state mRNA levels, while mutations in exon 5 had minimal effect.
  • Nuclear aprt mRNA levels were also reduced in mutant cells.
  • Nonsense-containing aprt mRNA decayed at similar rates to wild-type mRNA when transcription was globally inhibited with actinomycin D.
  • Nonsense-mediated destabilization of aprt mRNA was observed when transcription was specifically inhibited with tetracycline.
  • Low levels of nuclear nonsense-bearing aprt mRNA suggest a pre-export sensitivity to translation or translatability.
  • No accumulation of partially spliced aprt RNA indicated no splicing aberrations.

Conclusions:

  • Nonsense mutations in the aprt gene lead to reduced mRNA levels, primarily through post-transcriptional mechanisms.
  • Translation appears to play a role in the nuclear export of aprt mRNA.
  • The findings support a model where translation facilitates mRNA export, but the precise mechanism of nucleocytoplasmic communication remains to be determined.

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