Project 3

长核滞留的非编码RNA(IncRNAs)代表了一大类相对未被挖掘的RNAs，它们可能在基因调控和疾病病因学中发挥关键作用。一个主要的挑战是了解特定IncRNAs在细胞水平和生物体背景下的分子功能。该项目的长期目标是识别和鉴定在乳腺发育和乳腺癌的发生和发展中发挥关键作用的IncRNAs。在这里，提出了一系列目的，以剖析Malati的作用，Malati是一种丰富的IncRNA，定位于核斑点，并在相当数量的转移性乳腺癌中局部扩增。研究建议开发创新的功能丧失和功能获得的小鼠模型，结合细胞生物学方法来评估Malati在正常发育和乳腺癌起始和转移中的功能。将研究Malati水平的变化对组织组织的影响，并将通过下一代RNA测序分析来研究其对组织特定方式的选择性剪接的影响。补充细胞生物学研究将深入探讨Malati在核组织中所起的作用及其对富含核斑点的前mRNA剪接因子动态的影响。将使用RNA标签策略对Malati RNP进行纯化，并对其蛋白质组进行表征，以确定导致其核保留的蛋白质并提供对其功能的更多了解。在最终目标中，一系列新发现的在乳腺癌中被错误调控的IncRNAs将被优先考虑，并将选择其中几个进行功能分析，以确定它们所针对的基因和途径，并阐明它们在乳腺癌发生中的作用机制。总之，拟议的研究将为几个IncRNAs在正常发育和癌症中的作用提供重要的见解，并将为识别和表征一类新的、令人兴奋的潜在治疗靶点提供新的机会。

Long nuclear retained non-coding RNAs (IncRNAs) represent a large and relatively unmined class of RNAs that are likely to play critical roles in gene regulation and disease etiology. A major challenge is to understand the molecular functions of specific IncRNAs both at the cellular level and within the context of an organism. The long-term goal of this project is to identify and characterize IncRNAs that play a critical role in mammary gland development and the initiation and progression of breast cancer. Here, a series of Aims are presented to dissect out the role of Malati, an abundant IncRNA localized to nuclear speckles and focally amplified in a significant number of metastatic breast cancers. Studies are proposed to develop innovative loss-of-function and gain-of-function mouse models combined with cell biological approaches to assess the function of Malati in normal development and in breast cancer initiation and metastasis. The impact of alterations in the level of Malati on tissue organization will be examined, and its effect on alternative splicing in a tissue-specific manner will be pursued by next-generation RNA-sequencing analyses. Complementary cell biological studies will delve into the role that Malati plays in nuclear organization and its impact on the dynamics of pre-mRNA splicing factors enriched in nuclear speckles. The Malati RNP will be purified using an RNA-tagging strategy and its proteome will be characterized in order to identify proteins responsible for its nuclear retention and to provide additional insight into its function. In the final Aim a series of newly identified IncRNAs, that are misregulated in breast cancer, will be prioritized and several will be selected for functional analyses, to identify genes and pathways that they target, and to elucidate the mechanisms by which they contribute to breast cancer tumorigenesis. Together, the proposed studies will provide important insights into the role of several IncRNAs in normal development and cancer and will lead to new opportunities for the identification and characterization of a new and exciting class of potential therapeutic targets.