Analysis of the function, expression, and subcellular distribution of human tristetraprolin

Arthritis Rheum. 2002 May;46(5):1362-70. doi: 10.1002/art.10235.


Objective: The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNFalpha 3'-untranslated region (3'-UTR)-mediated expression. We also generated a specific polyclonal antibody against hTTP that enabled the examination of the subcellular distribution of hTTP and its RNA binding in vivo.

Methods: Transfection of reporter gene constructs were used to functionally characterize the role of hTTP in regulating TNFalpha expression in a 3'-UTR-dependent manner. An immunoprecipitation reverse transcription-polymerase chain reaction technique, immunoblotting, immunocytochemistry, and sucrose density fractionation were used to identify and localize hTTP.

Results: We found that hTTP interacted with human TNFalpha mRNA in the cytoplasm. The presence of the TNFalpha 3'-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNFalpha 3'-UTR adenine-uridine-rich element (ARE)-dependent manner. Immunoblotting and immunocytochemistry indicated that endogenous and transfected hTTP localized to the cytoplasm. Results of sucrose density fractionation studies were consistent with a polysomal location of hTTP. In rheumatoid synovium, hTTP expression was restricted to cells in the synovial lining layers.

Conclusion: Through the development of an antiserum specific for hTTP, we have been able to demonstrate that hTTP binds specifically to the TNFalpha 3'-UTR and reduces reporter gene expression in an ARE-specific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNFalpha. Understanding the posttranscriptional regulation of TNFalpha biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3' Untranslated Regions / physiology
  • Antibody Specificity
  • Arthritis, Rheumatoid / immunology
  • Base Sequence
  • Cell Line
  • DNA-Binding Proteins*
  • Gene Expression / physiology
  • Humans
  • Immediate-Early Proteins / analysis
  • Immediate-Early Proteins / genetics*
  • Immediate-Early Proteins / immunology*
  • Kidney / cytology
  • Luciferases / genetics
  • Molecular Sequence Data
  • Myeloid Cells / chemistry
  • RNA Processing, Post-Transcriptional / physiology
  • RNA, Messenger / analysis
  • Transfection
  • Tristetraprolin
  • Tumor Necrosis Factor-alpha / genetics*
  • Tumor Necrosis Factor-alpha / immunology


  • 3' Untranslated Regions
  • DNA-Binding Proteins
  • Immediate-Early Proteins
  • RNA, Messenger
  • Tristetraprolin
  • Tumor Necrosis Factor-alpha
  • ZFP36 protein, human
  • Luciferases