LncRNA Haunt在发育和干细胞分化中的功能和机制研究

Long noncoding RNAs (lncRNAs) have been hypothesized to be involved in diverse biological processes. However, their functions and biological significance are largely unknown. We have previously reported an opposing role for the DNA sequence and RNA transcripts of the lncRNA Haunt in regulating HOXA genes and embryonic stem cell differentiation. While the DNA sequence of Haunt functions as HOXA enhancers, Haunt RNA transcripts serve as a ‘brake’ to modulate its DNA enhancer activity, resulting in fine-tuned expression of HOXA genes during ESC differentiation (Yin et al., 2015. Cell Stem Cell). However, the detailed mechanisms by which Haunt RNA regulates HOXA gene expression and the physiological functions of Haunt are still unclear. We hypothesize that Haunt may exhibit its function by interacting with key regulatory proteins. To identify its interacting proteins in a systematic manner, we propose to use novel complementary biochemical approaches, including protein array hybridization, ChIRP and Csy4 RNA tagging following by mass spec sequencing. In addition, we will perform biochemical and structural study of the Haunt-protein complex to reveal the mechanisms of Haunt function. Meanwhile, we will study the physiological functions of Haunt by establishing a conditional knockout mouse. The results from the proposed work will provide an important framework for us to elaborate how Haunt functions to regulate local and distal chromatin structure and epigenetic landscapes of HOXA region through key RNA-protein interactions. Moreover, this work will establish novel experimental platforms and provide a new angle for mechanistic investigation of lncRNA functions, facilitating our overall understanding of the roles of noncoding RNAs in mediating transcriptional and epigenetic regulation in development and disease.

长链非编码RNA（lncRNA）在细胞命运决定过程中扮演重要角色。我们之前报导了lncRNA Haunt的转录本和DNA序列能拮抗性地参与调控HOXA基因簇的表达，并影响干细胞分化。但是，Haunt行使功能的分子机制及其在动物体内的生理功能还不清楚。本申请以Haunt为例，从分析lncRNA的结合蛋白入手，阐述lncRNA-蛋白结合的基本规律、作用和意义。我们将通过分析Haunt RNA-蛋白芯片杂交数据，建立优化染色质RNA富集（ChIRP-MS）和Csy4 RNA标签并结合质谱检测等手段，筛选与Haunt结合的功能性蛋白。并结合生物化学、结构解析和构建Haunt条件性敲除小鼠，揭示Haunt的生物学功能和具体机制。预期研究成果将为lncRNA的机制研究提供新思路和新手段，弥补lncRNA机制和生理功能研究的不足，有助于更好认识lncRNA在细胞命运决定、谱系分化等相关发育过程中的作用。

人类和鼠的基因组中存在大量结合于染色质的长链非编码RNA（lncRNA）。我们之前的工作报导了一种广泛存在的lncRNA顺式调控邻近基因时空表达的细微调控模式，也为lncRNA的功能预测提供依据（Cell Stem Cell, 2016）。另外，一些lncRNA（比如Haunt）的转录本和DNA序列，能同时拮抗性地促进和抑制同一靶基因的转录，从而精细控制靶基因的表达水平和干细胞分化的正常进行，为lncRNA机制研究提供新思路（Cell Stem Cell, 2015）。相比编码蛋白mRNA定位于细胞质，成百上千的非编码RNA结合在染色质上参与转录和表观遗传调控。然而lncRNA在染色质上结合的分子机制及其生理学功能尚不清楚。.本项目发现参与RNA剪接的U1小核糖核蛋白粒子（U1 snRNP），广泛调控非编码ncRNA在染色质上的富集和移动（Nature,2020）。我们开发了RNA 亚细胞定位元件测序的技术（REL-seq，mutREL-seq），对多种lncRNA 上的顺式调控元件进行了高通量分析。mutREL-seq高精度筛选发现 lncRNA上富集 U1 识别位点。与mRNA 相比，lncRNA 上富集U1 snRNA 识别位点 和5’剪接位点，但缺乏3’剪接位点，这导致lncRNA 具有高水平的与U1 snRNP 结合的能力。U1 snRNP持续结合在lncRNA转录本上，而不能通过RNA剪接过程释放，从而介导了lncRNA的染色质滞留。快速抑制U1 snRNA 或短时间降解SNRNP70 会显著降低lncRNA在染色质上的丰度，但对其转录水平没有明显影响。此外，RNA聚合酶Pol II进一步介导了ncRNA-U1 snRNP复合体在染色质上的移动。对于大多数低丰度、不稳定的ncRNA，它们只能靶向结合邻近的染色质区域（顺式作用）；而对于少数稳定和高丰度的lncRNA（如Malat1），U1 snRNP促进了其迁移到更多的靶基因区域（反式作用）。因此，U1 snRNP 以转录依赖的方式促进lncRNA 在染色质上的结合。这一发现为ncRNA反馈调控邻近转录和染色质结构提供了机制的解释，并拓宽了U1 snRNP的功能。