De novo protein synthesis is required for the activation-induced cytidine deaminase function in class-switch recombination.
Activation-induced cytidine deaminase (AID) is required for class-switch recombination (CSR), somatic hypermutation, and gene conversion of Ig genes. Although AID has sequence similarity to an RNA-editing enzyme Apobec-1, how AID functions in CSR and somatic hypermutation is unknown. Because involvement of RNA-editing but not DNA-editing in CSR requires de novo protein synthesis after AID expression, we examined whether protein synthesis inhibitors could block CSR in the presence of the AID activity. For this purpose we constructed AID fused with the hormone-binding domain of the estrogen receptor (AID-ER), which was introduced into AID-deficient spleen B cells. When such transfectants were treated with an estrogen analogue, 4-hydroxytamoxifen (OHT), CSR was induced within 1 h. Cycloheximide or puromycin drastically suppressed OHT-induced CSR in AID-ER expressing AID-/- B cells when added 1 h before OHT but not after OHT, suggesting that de novo protein synthesis is required for an event downstream to AID expression in CSR. The results lend the weight to RNA-editing hypothesis for the function of AID.
Doi
Tomomitsu
T
Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Kinoshita
Kazuo
K
Ikegawa
Masaya
M
Muramatsu
Masamichi
M
Honjo
Tasuku
T
D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D003513:Cycloheximide / Q000494:pharmacology; D003564:Cytidine Deaminase / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004965:Estrogen Antagonists / Q000494:pharmacology; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D017578:Immunoglobulin Class Switching; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology; D013347:Subcellular Fractions; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors
D004965:Estrogen Antagonists; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D011960:Receptors, Estrogen; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D011691:Puromycin; D012313:RNA; D004247:DNA; D003513:Cycloheximide; D003564:Cytidine Deaminase