AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C 3 U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.Affinity maturation of the humoral response proceeds by diversification of Ig genes (1, 2). Diversification requires AID 3 -initiated somatic hypermutation (SHM) within IgV regions and concomitant class switch recombination in Ig switch regions (S regions) (3). AID initiates SHM and class switch recombination by deaminating deoxycytidine residues during transcription of the Ig locus (4, 5). AID deaminates dC 3 dU on single-stranded DNA (ssDNA) and during the transcription of double-stranded DNA (6 -8), presumably on the non-transcribed strand. AID favors deamination in WRC (where W represents A/T, and R is A/G) hot spot motifs (7). The observation of strongly enhanced C 3 T mutations in variable regions and S regions, which conform to the mutational signature of AID (7), offers convincing evidence that AID is acting directly on the transcribed DNA. AID is located predominantly in the cytoplasm of activated B-cells (9) and has a nuclear localization signal motif in its N-terminal region and a nuclear export signal at its C terminus (10 -12). Deletion of the nuclear export signal results in nuclear accumulation (11, 12) but does not result in an increase in mutations in S regions (11). There must be a means to regulate the import of AID into the nucleus and then to direct its access to transcribed Ig loci. Presumably, AID is recruited to specific Ig regions by a variety of targeting mechanisms that could include cis-acting transcription factors, proteins that associate with AID, and regions of ssDNA formed by transcription bubbles (4, 5). There are coimmunoprecipitation (co-IP) data showing interactions between AID and potential recruiter molecules, including RNA polymerase II (13), a spliceosome-associated factor CTNNBL-1 (14), ssDNA-binding protein (RPA) (8, 15), and protein kinase A (16). However, it remains unclear how AID is targeted preferentially to actively transcribed Ig genes; no...
Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.Multicellular organisms maintain tissue homeostasis through response of their cells to extracellular signals that either promote their proliferation and differentiation or induce their death. Evidence is accumulating that extracellular stimuli, such as growth factors and cytokines, operate via complex signal transduction networks that ultimately control cellular fates (1); however, the complete molecular mechanisms need to be elucidated.One convenient system for studying the molecular mechanisms governing cell survival and death is spermatogenesis. It is mediated not only by cell proliferation and differentiation but also by programmed cell death or apoptosis, culminating in the production of spermatozoa. Apoptosis is necessary for eliminating unwanted cells and adjusting cell numbers in multicellular organisms to ensure tissue homeostasis.In the testis of the Japanese red-bellied newt, Cynops pyrrhogaster, primary spermatogonia proliferate through seven mitotic divisions, and then in the 8th generation the spermatogonia differentiate into primary spermatocytes and initiate meiosis in the spring when the ambient temperature is high; in contrast, in the autumn when the ambient temperature is low, the spermatogonia often undergo apoptosis in the 7th generation, resulting in the cessation of spermatogenesis (2). These findings suggest the existence of molecular mechanisms regulating the fate of 7th generation spermatogonia.We have shown in vitro and in vivo that the cellular fate in the spermatogonia is mainly regulated by changes in the endogenous levels of two peptide hormones secreted from the pituitary gland as follows: follicle-stimulating hormone (FSH) 2 th...
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