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AU-rich elements found in the 3'-untranslated regions of cytokine and proto-oncogene transcripts regulate mRNA degradation and function as binding sites for the mRNA-stabilizing protein HuA and the mRNA-destabilizing protein tristetraprolin. Experiments were performed to evaluate the expression of HuA and tristetraprolin in purified human T lymphocytes and to evaluate the ability of these proteins to recognize specific AU-rich sequences. HuA is a predominantly nuclear protein that can also be found in the cytoplasm of resting T lymphocytes. Within 1 h after stimulation of T lymphocytes with anti-T cell receptor antibodies or a combination of a phorbol myristate acetate and ionomycin, an increase in cytoplasmic HuA RNA-binding activity was observed. Although absent in resting cells, cytoplasmic tristetraprolin protein was detected 3-6 h following activation. HuA recognized specific AU-rich sequences found in c-jun or c-myc mRNA that were poorly recognized by tristetraprolin. In contrast, tristetraprolin recognized an AU-rich sequence in interleukin-2 mRNA that was poorly recognized by HuA. Both HuA and tristetraprolin, however, recognized AU-rich sequences from c-fos, interleukin-3, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor mRNA. HuA may transiently stabilize a subset of AU-rich element-containing transcripts following T lymphocyte activation, and tristetraprolin may subsequently mediate their degradation.

Publisher Summary This chapter focuses on HeLa nuclear extracts. The chapter presents a method that is used for the growth of HeLa cells. HeLa S3 cells (human epithelioid carcinoma, cervix) were obtained from ATCC stocks. The cells are adapted to grow in Joklik's MEM medium; containing 5% defined Bovine Calf Serum, supplemented with penicillin-streptomycin and NaHCO3 at 37°C. The cells were grown in suspension and stirred using magnetic bars at medium speed. All flasks should be tightly sealed in order to keep the carbon dioxide (CO2 ) at appropriate levels. HeLa cells are grown at an ideal density of 5–8 × 105/ml (minimum density should be 3 × 105/ml in 20 l of medium. HeLa cells double approximately every 24 h, and to keep a density of 5–8 × 105/ml, half of the volume of the cell suspension should be discarded daily and the same amount of fresh medium should be added until expansion is required. This chapter also discuses the extract preparation including: materials, methods, and nuclear extraction.