A mutually exclusive stem loop arrangement in roX2 RNA is essential for X chromosome regulation in Drosophila

The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX lncRNAs harbor several redundant domains to interact with the ubiquitin ligase MSL2 and the RNA helicase MLE for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By employing the uvCLAP methodology, which provides unprecedented information about RNA secondary structures in vivo, we identified the minimal functional unit of roX2 RNA. By using wild type and various MLE mutant derivatives including a catalytic inactive MLE derivative MLEGET we show that the minimal roX RNA contains two mutually exclusive stem-loops which exist in a peculiar structural arrangement: when one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. We show that this functional unit is necessary for dosage compensation as mutations that disrupt this formation lead to male lethality. Thus, we propose that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome. Overall design: UV crosslinking and affinity purification (uvCLAP) for MLE wildtype and 4 MLE mutants in Drosophila melanogaster.

X染色体是研究RNA-蛋白质相互作用（RNA-protein interactions）在表观遗传调控（epigenetic regulation）中发挥贡献的理想模型系统。在雄果蝇中，roX长链非编码RNA（long non-coding RNA, lncRNA）携带有多个冗余结构域，可与泛素连接酶MSL2（ubiquitin ligase MSL2）及RNA解旋酶MLE（RNA helicase MLE）相互作用，以实现X染色体的调控。然而，这些相互作用如何介导染色体铺展的分子机制仍尚不明确。
本研究采用uvCLAP技术（uvCLAP）——该技术可提供体内（in vivo）RNA二级结构（RNA secondary structures）的前所未有的详尽信息——成功鉴定出roX2 RNA的最小功能单元。通过使用野生型MLE及多种MLE突变体（包括催化失活的MLE变体MLEGET）开展实验，我们证实该最小roX RNA包含两个互斥的茎环结构（mutually exclusive stem-loops），二者以特殊的结构排布形式存在：当其中一个茎环被MLE解旋后，可形成另一种交替结构，这一过程可能将MLE捕获于这一持续形成的结构区域中。
我们证实该功能单元对于剂量补偿（dosage compensation）是必需的，破坏该结构形成的突变会导致雄性个体致死。据此，我们提出roX2长链非编码RNA包含一个依赖于MLE的亲和力开关，可介导MSL复合物（MSL complex）发生可逆相互作用，从而实现X染色体的剂量补偿。
整体实验设计：针对黑腹果蝇（Drosophila melanogaster）中的野生型MLE与4种MLE突变体开展紫外交联与亲和纯化（UV crosslinking and affinity purification, uvCLAP）实验。