Inhibition of tristetraprolin deadenylation by poly(A) binding protein.
Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.
Rowlett
Robert M
RM
Department of Medicine, University of Virginia, Charlottesville, VA, USA.
Chrestensen
Carol A
CA
Schroeder
Melanie J
MJ
Harp
Mary G
MG
Pelo
Jared W
JW
Shabanowitz
Jeffery
J
DeRose
Robert
R
Hunt
Donald F
DF
Sturgill
Thomas W
TW
Worthington
Mark T
MT
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005095:Exoribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010766:Phosphorylation; D039122:Poly(A)-Binding Protein I / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D051816:Tristetraprolin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D020413:3' Untranslated Regions; D004791:Enzyme Inhibitors; D005095:Exoribonucleases; D009355:Neomycin; D039122:Poly(A)-Binding Protein I; D012333:RNA, Messenger; D012262:Ribonucleoside Diphosphate Reductase; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; C497553:Zfp36 protein, mouse