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


CONCLUSIONS
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.
Brooks
Seth A
SA
Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire, USA.
Connolly
John E
JE
Diegel
Roger J
RJ
Fava
Roy A
RA
Rigby
William F C
WF
D020413:3' Untranslated Regions / Q000502:physiology; D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000276:immunology
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; C497552:ZFP36 protein, human; D008156:Luciferases