The Structure and Function of Perinucleolar Compartment

描述(申请人提供)：PNC在结构上与核仁相连，它们在细胞周期中与核仁一致地组装和拆解。它高度富含新合成的PolIII RNA，并依赖于PolIII的连续转录。PNC在有转移能力的细胞中形成，PNC的组装不仅仅是PNC成分过度表达的结果。在上一个资金周期(3.5年)中，以RNase MRP RNA(RMRP)为例，我们发现PNC含有一个新的含有RMRP的复合体。该复合体含有PTB、CUGBP、RMRP和其他未知因素，并且很大，在5-40%甘油梯度的底部造粒。利用高分辨率光学显微镜(OMX)，我们发现RMRP-PTB-CUGB在PNC处紧密共定位。然而，PNC上的RMRP不与新合成的RNA共定位，表明RNP可能不代表新生RMRP的转录后加工。此外，我们还发现PNC与未知的DNA基因座相关。这些发现导致了我们目前的工作模式。未知的RNP在特定的DNA位点上成核，很可能是PolIII基因(S)，并调节它们在含有PNC的细胞中的表达。这种调控可以是在DNA或RNA的水平上，也可以是两者兼而有之。我们提出了三个互补的目标来检验这一假说，并研究PNC的结构和功能。我们将确定新的RMRP-PTB-CUGBP复合体的组成以及该复合体与PNC和细胞活动的功能相关性。我们将鉴定与PNC相关的DNA基因座，并剖析新的RNP与这些基因座的表达之间的关系。这些实验结果不仅为进一步了解PNC的结构和功能与肿瘤的关系提供了必要的基础，而且还将揭示高度保守的RMRP的未知功能。

DESCRIPTION (provided by applicant): PNCs are structurally linked to the nucleoli, and they assemble and disassemble in concert with nucleoli through the cell cycle. It is highly enriched with newly synthesized pol III RNA and is dependent upon continuous pol III transcription. The PNC forms in metastatically capable cells and the assembly of the PNC is not simply the results of over expression of the PNC components. Over the last funding cycle (3.5 years), using RNase MRP RNA (RMRP) as an example, we have found that PNC contains a novel RMRP containing complex. The complex contains PTB, CUGBP, RMRP, and other unknown factors, and is large, pelleting at the bottom of a 5-40% glycerol gradient. Using a high resolution light microscopy (OMX), we found that RMRP-PTB-CUGB closely colocalize at the PNC. However, RMRP at the PNC does not colocalize with newly synthesized RNA, suggesting the RNP may not represent the post transcriptional processing of nascent RMRP. In addition, we found that the PNC is associated with unknown DNA loci. These findings lead to our current working model. Unknown RNPs become nucleated upon specific DNA loci, most likely a pol III gene(s), and regulates their expression in PNC containing cells. The regulation can be at the levels of DNA or RNA, or both. We propose three complementary aims to test this hypothesis and to investigate the structure and function of the PNC. We will identify the components of the novel RMRP-PTB-CUGBP complex and the functional relevance of this complex to the PNC and cellular activities. We will identify the PNC associated DNA loci and will dissect the relationship between the novel RNPs and the expression of the loci. The results of these experiments will not only provide the necessary foundation to the future understanding of the structure and function of the PNC and malignancy, but will also reveal uncharacterized function of highly conserved RMRP.