The Structure and Function of Perinucleolar Compartment

描述（由申请方提供）：PNC在结构上与核仁连接，并且它们在细胞周期中与核仁一起组装和分解。它高度富集新合成的pol III RNA，并依赖于连续的pol III转录。PNC在具有转移能力的细胞中形成，并且PNC的组装不仅仅是PNC组分过表达的结果。在上一个资助周期（3.5年）中，以RNase MRP RNA（RMRP）为例，我们发现PNC含有一种新的含RMRP的复合物。该复合物含有PTB、CUGBP、RMRP和其他未知因子，并且很大，在5-40%甘油梯度的底部沉淀。利用高分辨率光学显微镜（OMX），我们发现RMRP-PTB-CUGB紧密共定位在PNC。然而，RMRP在PNC不与新合成的RNA共定位，这表明RNP可能不代表新生RMRP的转录后加工。此外，我们发现PNC与未知的DNA位点相关。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基因座（最可能是pol III基因）上成核，并调节它们在含有PNC的细胞中的表达。调节可以在DNA或RNA水平，或两者。我们提出了三个互补的目的来验证这一假设，并调查PNC的结构和功能。我们将鉴定新型RMRP-PTB-CUGBP复合物的组分以及该复合物与PNC和细胞活性的功能相关性。我们将确定PNC相关的DNA位点，并将剖析新的RNP和基因座的表达之间的关系。这些实验结果不仅将为今后了解PNC的结构和功能以及恶性肿瘤提供必要的基础，而且还将揭示高度保守的RMRP的未知功能。 公共卫生相关性：核仁周围区室（PNC）是在能够转移的癌细胞中形成的核结构。为了了解PNC的结构和功能，我们建议鉴定其分子组分，并分析它们在正常和癌性条件下在PNC内外的功能。

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. PUBLIC HEALTH RELEVANCE: The perinucleolar compartment (PNC) is a nuclear structure forming in cancer cells that are capable of metastasis. To understand the structure and function of the PNC, we propose to identify its molecular components and analyze their function in and out of the PNC during normal and cancerous conditions.