The role of roX RNA for structure and function of the dosage compensation complex.

复杂的真核生物基因组编码大量的长非编码（lnc） rna。我们对基于染色质的lncRNA功能的分子机制知之甚少。我们将研究一种典型的多酶染色质调节剂，它的组装和功能绝对需要lncRNA。黑腹果蝇（Drosophila melanogaster）的剂量补偿复合体（DCC）由5个雄性特异性致死（male-specific-lethal， MSL）蛋白与非编码的roX RNA组合而成，形成一个具有特殊特性的调节复合体：DCC使雄性细胞中单个X染色体上大多数基因的转录加倍，以匹配雌性细胞中两个X的组合输出。虽然单独的蛋白质亚基可以相互结合并且在体外是活性酶，但roX的结合是细胞中DCC功能的重要要求。然而，RoX的功能是神秘的。DExH解旋酶MLE对roX的二级结构重塑启动了MSL蛋白与功能性DCC组装的关联。我们假设MSL蛋白通过与roX相关联而在DCC中获得其专用功能。我们的目的是阐明lncRNA roX整合到DCC中的分子基础，以了解DCC组装和活性对roX的依赖性。为此，我们将详细研究roX结合的机制，优化含有特定RNA的DCC的组装，并通过特异性蛋白质-RNA相互作用分析（包括交联、测序和质谱）表征roX- msl相互作用。我们的最终目标是比较存在和不存在特定RNA的MSL组装，并将结构变化与改进的MSL功能联系起来。我们的研究将揭示一种迷人的、高度进化的染色质调节剂。更重要的是，我们努力为新兴的lncRNA生物学领域揭示具有普遍相关性和广泛适用性的lncRNA功能调控原理。

Complex eukaryotic genomes encode abundant long, non-coding (lnc) RNAs. Our knowledge of the molecular mechanisms underlying chromatin-based lncRNA function is scarce.We will study a paradigmatic, multi-enzyme chromatin regulator, which absolutely requires lncRNA for assembly and function. The dosage compensation complex (DCC) of Drosophila melanogaster consists of five male-specific-lethal (MSL) proteins that assemble with non-coding roX RNA to form a regulatory complex with special properties: the DCC doubles the transcription of most genes on the single X chromosome in male cells to match the combined output of the two Xs in females.Although the protein subunits alone can associate with each other and are active enzymes in vitro, the association of roX is a vital requirement for DCC function in cells. RoX function, however, is mysterious. Secondary structure remodeling of roX by the DExH helicase MLE initiates association of MSL proteins towards assembly of a functional DCC. We hypothesize that the MSL proteins acquire their dedicated functionality within the DCC by association with roX. We aim to elucidate the molecular basis of lncRNA roX integration into the DCC to understand the roX dependency of DCC assembly and activity. To this end, we will study the mechanisms of roX incorporation in detail, optimise the assembly of DCC containing specific RNA and characterize the roX-MSL interactions through specific protein-RNA interaction assays (involving crosslinking, sequencing and mass spectrometry). Our final goal is to compare MSL assemblies in the presence and absence of specific RNA and to correlate structural changes with refined MSL functions.Our study will shed light on a fascinating, highly evolved chromatin regulator. More importantly, we strive to uncover regulatory principles of lncRNA function of general relevance and broad applicability for the emerging field of lncRNA biology.