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

Abstract

Objective. The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor ␣ (TNF␣) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNF␣ 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 TNF␣ expression in a 3-UTRdependent 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 TNF␣ mRNA in the cytoplasm. The presence of the TNF␣ 3-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNF␣ 3-UTR adenineuridine-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 TNF␣ 3-UTR and reduces reporter gene expression in an AREspecific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNF␣. Understanding the posttranscriptional regulation of TNF␣ biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis.

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

“…The NT condition is transfected with an equal amount of pcDNA 3.1 HisC, the backbone vector for the TTP expression construct. Consistent with previous results (19,(30)(31)(32), transfection of TTP into control shRNA cells results in an ∼25% decrease in TNF-39 UTR luciferase expression in resting cells (p , 0.01) (Fig. 5A) (30-32).…”

“…These studies employ pGL3-Control and TNF-39 UTR luciferase reporters, which differ only in the presence of the TNF-a 39 UTR, as well as several TTP expression constructs (5,19,(30)(31)(32)(33). Our hypothesis for these experiments was that TNF-a luciferase expression would be lower in the Cul4B shRNA cells compared with the control shRNA cells.…”

Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

“…Poly(A) RNA was purified using Oligotex beads (Qiagen). Isolation of a polysome-enriched fraction was performed as previously described (28). Cells were washed three times with 1ϫ phosphate-buffered saline and resuspended in 3.5 ml of Buffer A (10 mM Tris-HCl, pH 7.6, 1 mM KAc, 1.5 mM MgAc, 2 mM dithiothreitol, 1 mg/ml pepstatin A, 1 mg/ml leupeptin, 1 mM pefabloc).…”

Extracellular Signal-regulated Kinase Regulation of Tumor Necrosis Factor-α mRNA Nucleocytoplasmic Transport Requires TAP-NxT1 Binding and the AU-rich Element

“…Transient Transfections and Luciferase Assays-RAW 264.7 cell transfections were performed as previously described (37)(38)(39), using 1 g of luciferase or Renilla construct and 100 ng of each dominant negative vector, with any difference in total DNA made up by adding pcDNA3.1. The B-box N-Fusion protein luciferase assays were performed using 1 g of luciferase construct and 300 ng of N-Fusion protein vector, either N-TAP, N-NxT1, N-Aly, or N-UAP56 (36).…”