Forward Genetic Analysis of Human Nuclear Long Non-Coding RNAs

项目总结 某些人类核长非编码RNA(LncRNAs)代表着公认的有希望的靶点 治疗癌症、发育性和病毒性疾病；控制它们的水平有望提供 对各种人类疾病的治疗干预。然而，由于缺乏可靠的高吞吐量 量化核lncRNA水平的筛选技术，人类核incRNA生物发生的途径， 监管和监督一直难以向前推进基因鉴定。我们开发了一种方法 这使得第一次成功地对生物发生和监测的生物发生途径进行了前向遗传询问 人类细胞中的核lncRNA MALAT1。我们确定了MALAT1 3‘所需的人核复合体 MALAT1相关小细胞质RNA(MascRNA)的终末监测和所需成分 成熟度，以及许多需要进一步研究的候选基因。我们建议扩大这一范围 对作用于核蛋白的人类核通路进行全面的正向遗传鉴定 下列lncRNAs：(I)与癌症相关的lncRNA MALAT1，(Ii)多发性内分泌瘤转录本1(MENS- β)，其在胚胎干细胞和神经元分化中上调，和(Iii)多腺化 Kaposi肉瘤相关细胞裂解产生新病毒颗粒所需的核lncRNA PAN 疱疹病毒(KSHV)。这项研究的目的是确定作用于人类的途径和调节网络 并发现潜在的抗癌和抗病毒治疗的新靶点。

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.