Tsp57: a novel gene induced during a specific stage of spermatogenesis.
Recently, we described the identification of a novel protein, nuclear receptor-associated protein 80 (RAP80), which is highly expressed in spermatocytes and appears to have a role in regulating gene expression. To identify proteins interacting with this protein, we performed yeast two-hybrid screening using full-length RAP80 as bait. This screen identified one in-frame clone encoding a novel testis-specific protein (Tsp), referred to as Tsp57. Tsp57 encodes a basic protein with a mass of 56.8 kDa. The amino acid sequence of Tsp57 is highly conserved (87%) between mouse and human. The mouse and human Tsp57 genes map to chromosomes 9A1 and 11q21, respectively. Northern blot analysis showed that the expression of Tsp57 mRNA was highly restricted to the testis and temporally regulated during testicular development. Tsp57 mRNA was greatly induced between Day 21 and Day 25 of postnatal testicular development. In situ hybridization analysis demonstrated that the hybridization signal for Tsp57 mRNA was strongest in sections of seminiferous tubules at stages VI-VIII of spermatogenesis, consistent with the conclusion that Tsp57 is most highly expressed in round spermatids. Study of Tsp57 expression in several purified subpopulations of spermatogenic cells confirmed maximum levels of expression in round spermatids. Consistently, Tsp57 expression was absent in testes from vitamin A-deficient mice, which do not have any round spermatids, and was reduced in RARalpha null mice, which have lowered numbers of round spermatids in their testes. These results indicate the possibility that Tsp57 protein plays a role in the postmeiotic phase of germ cell differentiation. Tsp57 contains two putative nuclear localization signals: NLS1 and NLS2. Examination of the cellular localization showed that the green fluorescent protein-Tsp57 fusion protein localized to both cytoplasm and nucleus. After deletion of NLS1 but not NLS2, Tsp57 localized solely to the cytoplasm, indicating a role for NLS1 in the nuclear localization of Tsp57. The localization suggests a nuclear function for Tsp57. Pull-down analysis demonstrated that Tsp57 and RAP80 form a complex in intact cells.
Kim
Yong-Sik
YS
Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Nakanishi
Gen
G
Oudes
Asa J
AJ
Kim
Kwan Hee
KH
Wang
Hang
H
Kilpatrick
Daniel L
DL
Jetten
Anton M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins; D033481:Chromosomes, Mammalian; D004268:DNA-Binding Proteins; D005260:Female; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D056488:Histone Chaperones; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D018168:Receptors, Retinoic Acid / Q000235:genetics; D000072482:Retinoic Acid Receptor alpha; D013087:Spermatids / Q000502:physiology; D013091:Spermatogenesis / Q000502:physiology*; D013737:Testis / Q000166:cytology / Q000196:embryology / Q000502:physiology; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D008164:Luminescent Proteins; D009687:Nuclear Proteins; C000607071:RARA protein, human; D012333:RNA, Messenger; C000607072:Rara protein, mouse; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; C482107:Tsp57 protein, mouse; C466234:UIMC1 protein, human; D049452:Green Fluorescent Proteins