Tristetraprolin recruits functional mRNA decay complexes to ARE sequences

Heidi H. Hau, Richard J. Walsh, Rachel L. Ogilvie, Darlisha A. Williams, Cavan S. Reilly, Paul R. Bohjanen

Research output: Contribution to journalArticlepeer-review

79 Scopus citations

Abstract

AU-rich elements (AREs) in the 3′ untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3′ UTR promoted TTP-dependent mRNA decay when it was inserted into the 3′ UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5′-3′ exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript.

Original languageEnglish (US)
Pages (from-to)1477-1492
Number of pages16
JournalJournal of Cellular Biochemistry
Volume100
Issue number6
DOIs
StatePublished - Apr 15 2007

Keywords

  • AU-rich elements
  • Deadenylation
  • Exosome
  • PM-scl75
  • Tristetraprolin
  • Xrn1
  • mRNA decay
  • mRNA degradation

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