Tristetraprolin recruits functional mRNA decay complexes to ARE sequences.
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.
Hau
Heidi H
HH
Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Walsh
Richard J
RJ
Ogilvie
Rachel L
RL
Williams
Darlisha A
DA
Reilly
Cavan S
CS
Bohjanen
Paul R
PR
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002474:Cell-Free System / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D011485:Protein Binding; D020871:RNA Stability; D014162:Transfection; D051816:Tristetraprolin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D020413:3' Untranslated Regions; D007376:Interleukin-2; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor