Nonsense codons trigger an RNA partitioning shift

J Biol Chem. 2009 Feb 13;284(7):4062-72. doi: 10.1074/jbc.M805193200. Epub 2008 Dec 17.

Abstract

T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism*
  • Codon, Nonsense / genetics
  • Codon, Nonsense / metabolism*
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism
  • Down-Regulation / physiology
  • Eukaryotic Initiation Factor-4A
  • Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / physiology*
  • HeLa Cells
  • Humans
  • Kinetics
  • Pol1 Transcription Initiation Complex Proteins / genetics
  • Pol1 Transcription Initiation Complex Proteins / metabolism
  • RNA Interference
  • RNA Stability / physiology*
  • RNA, Messenger / biosynthesis*
  • RNA, Messenger / genetics
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Receptors, Antigen, T-Cell, alpha-beta / biosynthesis*
  • Receptors, Antigen, T-Cell, alpha-beta / genetics

Substances

  • Codon, Nonsense
  • Pol1 Transcription Initiation Complex Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Receptors, Antigen, T-Cell, alpha-beta
  • UPF3B protein, human
  • transcription factor UBF
  • Eukaryotic Initiation Factor-4A
  • EIF4A3 protein, human
  • DEAD-box RNA Helicases