Abstract
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.
Highlights
Multicellular organisms maintain tissue homeostasis through response of their cells to extracellular signals that either promote their proliferation and differentiation or induce their death
This study demonstrated the following: 1) a putative glycinerich RNA-binding protein disappears in the cytoplasm of 7th generation spermatogonia after prolactin exposure; 2) this loss is implicated in the induction of apoptosis and in the suppression of translation for the newt orthologue of programmed cell death protein 4 mRNA, the 3Ј-untranslated region (UTR) that interacts with newt RNA-binding protein (nRBP) protein; and 3) overexpression of nRBP protects intact cells from apoptosis and accelerates the translation of the chimeric green fluorescent protein (GFP)-newt programmed cell death protein 4 (nPdcd4) 3ЈUTR mRNA by increasing its stability
Injection of follicle-stimulating hormone (FSH) into newts had little effect on the endogenous expression of nPdcd4 mRNA and protein in the testis (Fig. 7, D and E). We showed that both mRNA and protein for nRBP are reduced in 7th generation spermatogonia during prolactin-dependent apoptosis, whereas nPdcd4 mRNA, which associates with endogenous nRBP protein, is transiently induced but its protein is reduced
Summary
Reagents and Animals—The antigen peptide (FVSEGDGGRLKPESY) for an antibody to human Pdcd (Rockland) and the PCR primers were produced in TORAY Research Center Co., Ltd., and Hokkaido System Science Co., Ltd., Japan, respectively. The frequency of apoptosis was expressed as a using cytoplasmic extracts (0.5 mg of protein) prepared by CEB containing RNase inhibitor and 0.1% Nonidet P-40 and lacking DTT from the transfectants with a combination of pME18snRBP and either pEGFP-nPdcd4 –3ЈUTR-FL, -1208, -814, -380, or the GFP vector. Total RNA extracted from the cells 12 h after transfection was reversetranscribed, and PCR was performed for 35 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 2 min for the chimeric gene mRNA, GFP/nPdcd4 –3ЈUTR-FL (2.2 kbp), -1208 (1.9 kbp), -814 (1.5 kbp), and -380 (1.1 kbp), with the GFP forward primer and each reverse primer of the nPdcd full-length and mutant 3ЈUTR. Signals detected in the Western blotting and RT-PCR were quantified by densitometry using ImageJ software
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