Life, Death, and Function: The Primate-Specific Long Non-Coding RNA Transcriptome

人类基因组计划的完成留下了一个持久的谜团：为什么蛋白质编码基因只占基因组序列的一小部分？基因组的其余部分有哪些功能元件？近十年后，在2012年，我参与的ENCODE（DNA元素百科全书）联盟揭示了人类基因组中异常丰富的长非编码RNA（lncRNA）基因（Derrien et al 2012）。这一成就部分建立在我之前的工作基础上（Jia et al 2010）。与microRNA相反，lncRNA通过不同的和异质的机制起作用，作为基因表达的正调节剂和负调节剂。在lncRNA的特性中，它们的低物种间保守性特别有趣：近5000个 人类lncRNA在灵长类动物之外并不保守。这是有趣和重要的，因为传统上，在进化中保守的蛋白质编码基因被认为是负责正常细胞过程和疾病中的大多数功能结果。然而，进化谱系特异性的生物和病理反应和机制越来越清楚。非保守lncRNA在人类疾病中具有谱系特异性和物种特异性功能吗？为了研究这一点，我将研究进化上非保守的人类lncRNA在生物学和临床相关系统中的功能：人类MCF 7细胞，一种已建立的雌激素受体α阳性乳腺癌体外模型。为了生成初步数据，我利用我的定制人类lncRNA微阵列（Lipovich等2012）来询问MCF 7细胞中lncRNA的雌激素反应性。我鉴定了127个雌激素反应性lncRNA，通过RNAi和过表达分析了18个，然后进行了一组6个表型测定。雌激素诱导的灵长类特异性lncRNA的敲低和雌激素抑制的灵长类特异性lncRNA的过表达降低了细胞活力和增殖，并且在一些情况下导致细胞死亡。这一发现促使我提出了一个中心假设：某些灵长类特异性lncRNA使人类细胞沿着增殖-增殖轴移动。在这个项目中，我将继续这一假设，并在此基础上进行扩展。我将把这六个功能分析扩展到所有127个导联。我将使用第二代和第三代RNAseq代替微阵列来估算完整的MCF 7雌激素响应性lncRNAome。我将从RNAseq数据中识别新型灵长类动物特异性lncRNA，并将它们置于我优化的系统扰动和表型测定工作流程中。已经在ENCODE Consortium的框架内显示了一些lncRNA的异位翻译（B nfai et al 2012），我还将测试灵长类特异性功能性lncRNA是否直接作为RNA发挥作用，而不是直接作为RNA发挥作用。 通过翻译的肽。研究结果将探讨一个有趣的新问题，有可能引发癌症生物学的范式转变：人类癌症在某种程度上是由非保守lncRNA引起的灵长类特异性疾病吗？这一提议的实施将导致首次将细胞功能保守无偏的高通量分配给与癌症治疗高度相关的主要核激素受体途径中的灵长类动物特异性lncRNA。

DESCRIPTION (provided by applicant): The completion of the Human Genome Project left an enduring mystery: Why do protein-coding genes account for only a tiny fraction of the genome sequence? What functional elements reside in the rest of the genome? Nearly a decade later, in 2012, the ENCODE (Encyclopedia of DNA Elements) Consortium, in which I participate, revealed the extraordinary abundance of long non-coding RNA (lncRNA) genes in the human genome (Derrien et al 2012). This achievement builds, in part, on my prior work (Jia et al 2010). In contrast to microRNAs, lncRNAs act through diverse and heterogeneous mechanisms, as both positive and negative regulators of gene expression. Among the properties of lncRNAs, their low interspecies conservation is particularly intriguing: nearly 5,000 human lncRNAs are not conserved beyond primates. This is interesting and important because, traditionally, protein-coding genes conserved in evolution were thought to be responsible for most functional outcomes in normal cellular processes and in disease. However, evolutionary lineage-specific biological and pathological responses and mechanisms are increasingly clear. Do non-conserved lncRNAs have lineage- and species-specific functions in human disease? To investigate this, I will examine the functions of evolutionarily non-conserved human lncRNAs in a biologically and clinically relevant system: human MCF7 cells, an established in-vitro model of estrogen receptor alpha positive breast cancer. To generate preliminary data, I utilized my custom human lncRNA microarray (Lipovich et al 2012) to interrogate lncRNAs for estrogen responsiveness in MCF7 cells. I identified 127 estrogen-responsive lncRNAs, analyzing 18 by RNAi and overexpression, followed by a panel of six phenotypic assays. Knockdown of estrogen- induced, and overexpression of estrogen-repressed, primate-specific lncRNAs reduced cell viability and proliferation, and in several cases caused cell death. This finding prompted my central hypothesis: certain primate-specific lncRNAs shift human cells along the apoptosis-proliferation axis. In this project, I will pursue this hypothesis and expand upon it. I ill extend the six functional assays to all 127 leads. I will employ second- and third-generation RNAseq, instead of microarrays, to impute the complete MCF7 estrogen- responsive lncRNAome. I will identify novel primate-specific lncRNAs from RNAseq data and subject them to my optimized workflow of system perturbations and phenotypic assays. Having shown ectopic translation of some lncRNAs within the framework of the ENCODE Consortium (B¿nfai et al 2012), I will also test whether primate-specific functional lncRNAs act directly as RNAs, not via translated peptides. The results will approach a fascinating new question with the potential to initiate a paradigm shift in cancer biology: Is human cancer, to an extent, a primate-specific disease caused by non-conserved lncRNAs? Implementation of this proposal will result in the first ever conservation-unbiased, high-throughput assignment of cellular functions to primate-specific lncRNAs in a major nuclear hormone receptor pathway that is highly relevant to cancer therapeutics.