Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a.
Molecular cloning and expression of bovine nucleoplasmin 2 (NPM2): a maternal effect gene regulated by miR-181a.
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DOI：
10.1186/1477-7827-9-40
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发表时间：
2011-03-29
期刊：
影响因子：
--
通讯作者：
Yao J
中科院分区：
文献类型：
--
作者：
Lingenfelter BM;Tripurani SK;Tejomurtula J;Smith GW;Yao J
核浆蛋白 2 (NPM2) 是一种卵母细胞特异性核蛋白，对于核和核仁组织以及早期胚胎发育至关重要。本研究的目的是克隆牛 NPM2 基因，确定其在卵母细胞发育和早期胚胎发生过程中的时间表达，并评估 miRNA-181a 在调节其表达中的潜在作用。使用根据人和小鼠 NPM2 cDNA 序列的保守区域设计的引物，从牛胎儿卵巢中扩增出 329 bp 的 cDNA 片段。进行RACE实验以获得牛NPM2 cDNA的5'和3'末端。使用实时PCR和蛋白质印迹分析来检测牛NPM2在卵母细胞和早期胚胎中的表达。在 Hela 细胞中进行牛 NPM2 和 miRNA-181a 的共表达以确定牛 NPM2 的表达是否受 miRNA-181a 调节。牛 NPM2 cDNA 长度为 851 bp，编码 200 个氨基酸的蛋白质。该蛋白含有保守的二分核定位序列，与小鼠和人 NPM2 分别具有 53% 和 62% 的同一性。牛 NPM2 mRNA 的表达仅限于卵巢。 NPM2 mRNA 在 GV 和 MII 期卵母细胞中丰富，在早期卵裂期胚胎中减少，在桑葚和囊胚期胚胎中几乎检测不到。同样，NPM2 蛋白在卵母细胞和早期胚胎中表达较高，但在囊胚中表达极低。从持续性优势卵泡中分离的卵母细胞中，NPM2 mRNA 的丰度显着低于从生长优势卵泡中分离的卵母细胞（P < 0.05）。鉴定出 NPM2 转录物 3'UTR 中的 miR-181a 结合位点。转染实验表明，与不含 miR-181a 的对照细胞相比，表达 miR-181a 的 Hela 细胞中的牛 NPM2 蛋白表达降低，表明 NPM2 的翻译受到 miR-181a 的抑制。我们的数据表明，牛 NPM2 的表达在早期胚胎发生过程中受到暂时调节，miR-181a 可能在其调节中发挥作用。
Nucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression. A 329 bp cDNA fragment was amplified from bovine fetal ovary using primers designed based on the conserved regions of the human and mouse NPM2 cDNA sequences. RACE experiments were performed to obtain the 5' and 3' ends of the bovine NPM2 cDNA. Real time PCR and Western blot analysis were used to examine the expression of bovine NPM2 in oocytes and early embryos. Co-expression of bovine NPM2 and miRNA-181a in Hela cells was performed to determine if expression of bovine NPM2 is regulated by miRNA-181a. The bovine NPM2 cDNA is 851 bp in length encoding a protein of 200 amino acids. The protein contains the conserved bipartite nuclear localization sequence and shows 53% and 62% identity with mouse and human NPM2, respectively. Expression of bovine NPM2 mRNA is restricted to ovaries. NPM2 mRNA is abundant in GV and MII stage oocytes, decreases in early cleavage stage embryos, and barely detectable in morula and blastocyst stage embryos. Similarly, expression of NPM2 protein is high in oocytes and early embryos but extremely low in blastocysts. The abundance of NPM2 mRNA is significantly lower in oocytes isolated from persistent versus growing dominant follicles (P < 0.05). A miR-181a binding site in the 3'UTR of the NPM2 transcript was identified. Transfection experiments showed that bovine NPM2 protein expression is reduced in Hela cells expressing miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a. Our data suggest that expression of bovine NPM2 is temporally regulated during early embryogenesis and miR-181a may play a role in its regulation.
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影响因子：
14.9
作者：
Rusinov, V;Baev, V;Minkov, IN;Tabler, M
通讯作者：
Tabler, M
影响因子：
2.5
作者：
Bettegowda, A;Patel, OV;Smith, GW
通讯作者：
Smith, GW
影响因子：
56.9
作者：
Giraldez, AJ;Mishima, Y;Schier, AF
通讯作者：
Schier, AF
DOI：
10.1530/jrf.0.1160293
发表时间：
1999-07-01
期刊：
JOURNAL OF REPRODUCTION AND FERTILITY
影响因子：
--
作者：
Mihm, M;Curran, N;Roche, JF
通讯作者：
Roche, JF
DOI：
10.1530/jrf.0.1060039
发表时间：
1996-01-01
期刊：
JOURNAL OF REPRODUCTION AND FERTILITY
影响因子：
--
作者：
Revah, I;Butler, WR
通讯作者：
Butler, WR