Department of Applied Molecular Biosciences, Nagoya University, Japan.
Cell Biochem Funct. 2010 Jun;28(4):321-8. doi: 10.1002/cbf.1662.
Involution of the mammary gland is a regressive phase that occurs after lactation, and requires reprogramming of gene expression for the tissue to return to a pre-pregnant state. Although the transcriptome of the mammary gland demonstrates complex changes at the mRNA level, the molecular mechanisms governing post-transcriptional control remain obscure. In the present study, we isolated cytoplasmic mRNA-protein complexes (mRNPs) from the mouse mammary gland at the early involution stage using discontinuous sucrose density ultracentrifugation. mRNPs including untranslated mRNAs were then purified with oligo(dT) immobilized on cellulose or paramagnetic beads. Proteins in the purified complexes were subjected to one/two-dimensional gel electrophoresis followed by mass spectrometry. This identified heterogeneous nuclear ribonucleoprotein A/B (Hnrpab), along with three other heterogeneous nuclear ribonucleoproteins. Hnrpab in the mRNPs reproducibly increased within 48 h after weaning and became one of the major components. When a vector expressing Hnrpab was transfected into two different cell lines, their growth was suppressed, demonstrating that this protein has cytostatic activity. These results suggest that early involution can be used as a model for understanding the mechanism of post-transcriptional control of gene expression, responsible for modulation of cell function.
乳腺的退化是哺乳期后的一个退化阶段，需要重新编程基因表达以使组织恢复到怀孕前的状态。尽管乳腺的转录组在 mRNA 水平上表现出复杂的变化，但调控转录后控制的分子机制仍不清楚。在本研究中，我们使用不连续蔗糖密度超速离心法从小鼠乳腺的早期退化阶段分离细胞质 mRNA-蛋白复合物（mRNPs）。然后，用固定在纤维素或顺磁珠上的寡聚（dT）纯化包含未翻译 mRNA 的 mRNPs。纯化复合物中的蛋白质进行一维/二维凝胶电泳，然后进行质谱分析。这鉴定出异质核核糖核蛋白 A/B（Hnrpab）以及另外三种异质核核糖核蛋白。Hnrpab 在断奶后 48 小时内重复增加，成为主要成分之一。当表达 Hnrpab 的载体转染到两种不同的细胞系中时，它们的生长受到抑制，表明该蛋白具有细胞抑制活性。这些结果表明，早期退化可以作为理解基因表达转录后调控机制的模型，负责调节细胞功能。
