Detection of early gene expression changes during activation of human primary lymphocytes by in vitro synthesis of proteins from polysome-associated mRNAs.
The rapid increase in protein synthesis during the mitogenic stimulation of human peripheral blood lymphocyte is the result of global and specific translational control mechanisms. To study some of these mechanisms, we examined the in vitro translatability of mRNAs associated with the polyribosome fraction. Polyribosome fractions were isolated from lymphocytes after activation with ionomycin and the phorbol ester PMA. The associated PAmRNAs were translated in the presence of mRNA-depleted rabbit reticulocyte lysate and [(35)S]Met, and the protein products were analyzed by SDS--PAGE and autoradiography. There was little synthesis of protein from the PAmRNAs isolated from unactivated T cells, but the PAmRNAs isolated from activated T cells showed a rapid increase in translatability. Translation of the PAmRNAs was sensitive to edeine and m7GTP, suggesting their cap-dependent translation. With activation, the majority of proteins showed increasing in vitro translation, but two proteins, p72 and p33, were found to have increased synthesis within 30 min, which decreased in 1 h. Transcription inhibitors were used to ascertain if regulation of their expression was transcriptional or translational. To identify these proteins, we used biotinylated lysine during the in vitro translation reaction, and we extracted the biotinylated protein by using streptavidin magnetic beads. The protein product was analyzed by mass spectrometry. p33 was identified as a prohibitin-like protein (BAP37), but the identification of p72 was not found in the databases. The distinct up-regulation and down-regulation of their protein expression suggest their tightly controlled regulation during early T cell activation.
Molecular Hematology Branch, Section on Protein and RNA Biosynthesis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. smiyamot@ucdavis.edu
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Miyamoto
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Qin
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Safer
D000818:Animals; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011132:Polyribosomes / Q000378:metabolism*; D000091402:Prohibitins; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012097:Repressor Proteins; D012156:Reticulocytes / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation
D019384:Nucleic Acid Synthesis Inhibitors; C000717331:PHB2 protein, human; D000091402:Prohibitins; D011506:Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D019809:Streptavidin; D008239:Lysine