Forward Genetic Analysis of Human Nuclear Long Non-Coding RNAs

项目摘要 某些人核长非编码RNA（lncRNA）代表了用于靶向肿瘤细胞的成熟的有希望的靶标。 治疗癌症，发育和病毒性疾病;操纵它们的水平，预计将提供 对各种人类疾病的治疗干预。然而，由于缺乏可靠的高通量 量化核lncRNA水平的筛选技术，核lncRNA生物合成的人类途径， 监管和监督一直难以向前遗传鉴定。我们开发了一种方法 这使得第一次成功地对生物发生的途径进行正向遗传询问和监测成为可能。 人类细胞中的核lncRNA MALAT 1。我们鉴定了MALAT 1 3′所需的人类核复合物， MALAT 1相关小细胞质RNA（mascRNA）的末端监视和所需组分 成熟，以及许多候选基因，需要进一步研究。我们建议扩大这一点， 方法，并进行全面的人类核途径的前向遗传鉴定作用于 以下lncRNA：（i）癌症相关lncRNAMALAT 1，（ii）多发性内分泌瘤转录物1（MEN-1）， β），其在胚胎干细胞和神经元分化后上调，和（iii）聚腺苷酸化 核lncRNA PAN，卡波西肉瘤相关的细胞裂解产生新的病毒颗粒所需的 疱疹病毒（KSHV）。这项研究的目的是确定人类的途径和调控网络， 核lncRNA和发现潜在的抗癌和抗病毒疗法的新靶点。

PROJECT SUMMARY Certain human nuclear long non-coding RNAs (lncRNAs) represent well-established promising targets for the treatment of cancer, developmental and viral diseases; manipulating their levels is expected to provide therapeutic interventions for various human disorders. However, due to the lack of a reliable high-throughput screening technology to quantify nuclear lncRNA levels, human pathways of nuclear lncRNA biogenesis, regulation, and surveillance have been refractory to forward genetic identification. We developed an approach that enables the first successful forward genetic interrogation of the pathways of biogenesis and surveillance of the nuclear lncRNA MALAT1 in human cells. We identified human nuclear complexes required for MALAT1 3′ end surveillance and components required for MALAT1-associated small cytoplasmic RNA (mascRNA) maturation, as well as numerous candidate genes that require further investigation. We propose to expand this approach and perform comprehensive forward genetic identification of human nuclear pathways acting on the following lncRNAs: (i) cancer-associated lncRNA MALAT1, (ii) multiple endocrine neoplasia transcript 1 (MEN- β), which is up-regulated upon embryonic stem cell and neuronal differentiation, and (iii) polyadenylated nuclear lncRNA PAN, required for lytic production of new viral particles by the Kaposi’s sarcoma-associated herpesvirus (KSHV). This study is aimed to identify human pathways and regulatory networks acting on nuclear lncRNAs and uncover new targets for potential anti-cancer and anti-viral therapies.