ORGANIZATION OF SMALL NUCLEOLAR RIBONUCLEOPROTEINS (SNORNPS) BY FLUORESCENCE IN-SITU HYBRIDIZATION AND IMMUNOCYTOCHEMISTRY
ORGANIZATION OF SMALL NUCLEOLAR RIBONUCLEOPROTEINS (SNORNPS) BY FLUORESCENCE IN-SITU HYBRIDIZATION AND IMMUNOCYTOCHEMISTRY
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DOI：
10.1091/mbc.5.12.1289
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发表时间：
1994-12-01
影响因子：
3.3
通讯作者：
WARD, DC
中科院分区：
文献类型：
--
作者：
MATERA, AG;TYCOWSKI, KT;WARD, DC
在HeLa细胞中，使用反义DNA和2 '-OMe RNA寡核苷酸研究了U3、U8和U13小核仁核糖核蛋白（snoRNP）的组织。合成了与靶向RNase H降解完整RNP颗粒的脱氧核苷酸对应的寡聚体，并用于荧光原位杂交。U3和U13分布在整个核仁中，并与抗原纤维蛋白抗体共定位。然而，U8在核仁中心附近组织成离散的环状结构，并围绕用抗RNA聚合酶I和抗UBF/NOR-90抗体可视化的明亮点状区域。在去致密的核仁中，出现了U8 RNA的较小环状结构的项链。一个模型的招聘U8（和可能的其他加工因素）的rRNA转录的网站进行了讨论。与有丝分裂细胞的杂交显示，与pol I和NOR-90不同，U8在有丝分裂期间分散到细胞质中。U8的亚核仁组织与其在前rRNA加工的早期中间步骤中的参与是一致的。与此相反，更分散的核仁内分布的U3同意其假定参与rRNA成熟的早期和晚期步骤。这些研究说明了通过将小核RNP的体外活性与它们的原位位置相关联来绘制核仁内的功能结构域的可行性。
The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.