M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

我们此前开发了一种用于分离编码有丝分裂期磷酸化蛋白（M phase phosphoproteins，MPPs）的互补DNA（complementary DNA，cDNAs）的新型技术。在本文所述研究中，我们对所鉴定的10种新型蛋白之一的MPP10展开进一步表征，以探究其潜在的核仁功能。通过细胞分级分离实验，我们发现几乎所有MPP10均存在于分离得到的核仁中。免疫荧光检测结果显示，在间期细胞内，MPP10与核仁纤维蛋白（fibrillarin）及其他已知核仁蛋白共定位，但在以纤维蛋白或仅存在于卷曲小体中的p80线圈蛋白（p80 coilin）染色的卷曲小体中未检测到MPP10。经放线菌素D（actinomycin D）处理将核仁拆分为纤维状与颗粒状结构域后，几乎所有MPP10均定位于纤维帽区域，该区域包含参与核糖体RNA（ribosomal RNA，rRNA）加工的蛋白。在细胞周期的早中期有丝分裂阶段，MPP10与纤维蛋白共定位于染色体表面。在有丝分裂末期，MPP10分布于类似核仁衍生小体与前核仁小体的细胞结构中。不过其中部分小体并不含有此前被报道为核仁衍生小体和前核仁小体组分的纤维蛋白，且大部分MPP10抵达核仁的时间晚于纤维蛋白。为进一步解析MPP10的特性，我们从细胞超声裂解物中免疫沉淀了MPP10。所得沉淀物中含有U3小核仁RNA（small nucleolar RNA，snoRNA），但未检测到其他大量的C/D框小核仁RNA。MPP10与U3 snoRNA的这种结合可耐受400 mM盐浓度，这表明MPP10是人类U3小核仁核糖核蛋白复合物的组分之一。