Non-canonical role of roX RNA in X-chromosome targeting

黑腹果蝇的X染色体剂量补偿过程确保了两性基因组的表达相似。雄性果蝇只有在其单X染色体上的基因转录在两倍范围内得到提升时才能存活。探索这一高度进化过程的机制基础将继续揭示基因组调控的基本原理。雄性果蝇X染色体基因的激活是通过剂量补偿复合物（DCC）实现的，DCC是一种由5种雄性特异性致死（MSL）蛋白和长链非编码roX RNA组成的多酶组装体。DCC与X染色体的初始高度选择性结合是忠实剂量补偿的关键。这一过程的损伤会导致染色体不加区分地结合，这对受影响的雄性果蝇是致命的。roX RNA和介导lncRNA组装到DCC的解旋酶MLE的作用尚不清楚。DCC实际上是为数不多的染色质结合lncRNPs之一，其所有RNA和蛋白质亚基都是已知的，因此为详细研究lncRNA的功能提供了巨大的机会。我们最近发现了roX2 RNA在X染色体选择性中的重要作用。奇怪的是，这种rna最直观的功能似乎不适用于roX。RoX显然不通过三螺旋或r环的形成读出DANN序列。RoX也不能作为支架来促进复合物的形成，因为MSL蛋白在没有RNA的情况下可以从活性复合物中分离出来。我们还发现，在roX存在的情况下，DCC与X染色体的特异性结合与采用紧密结合模式相关，在FRAP实验中记录了异常长的停留时间。我们试图揭示新的原理和潜在的分子机制，通过roX rna促进MSL复合物与X染色体的强大和排他性结合，以及DCC在X染色体上的“紧密结合”状态。

The process of X chromosome dosage compensation in Drosophila melanogaster assures that genomes of both sexes are expressed similarly. Male flies can only live if the transcription of genes on their single X chromosome is boosted in the two-fold fange. Exploring the mechanistic underpinnings of this highly evolved process continues to uncover fundamental principles of genome regulation. The activation of X chromosomal genes in male flies is achieved by the dosage compensation complex (DCC), a multi-enzyme assembly consisting of five male-specific-lethal (MSL) proteins and long, non-coding roX RNA. The initial, highly selective binding of the DCC to the X chromosome is key to faithful dosage compensation. Impairment of this process leads to indiscriminate chromosome binding, which is lethal for affected male flies. The role of roX RNA and the helicase MLE that mediates assembly of this lncRNA into the DCC is poorly understood. The DCC is actually one of the very few chromatin-bound lncRNPs, for which all RNA and protein subunits are known, thus providing a huge opportunity to investigate the function of a lncRNA in detail. We recently discovered an important role for roX2 RNA in X chromosome selectivity. Curiously, the most intuitive functions for such RNAs appear not to apply to roX. RoX apparently does not read out DANN sequence by triple-helix or R-loop formation. RoX also does not function as a scaffold to facilitate complex formation, since the MSL proteins can from active complexes in the absence of RNA. We also found that the specific binding of the DCC to the X chromosome in the presence of roX correlated with the adoption of a tight-binding mode, documented by unusually long residence times in FRAP experiments. We seek to uncover novel principles and underlying molecular mechanism, through which roX RNAs promote the robust and exclusive binding of the MSL complex to the X chromosome, and which underlie the ‘tight-binding’ state of the DCC on the X chromosome.