Deciphering the molecular role of Spenito, a novel component of the m6A methyltransferase complex

表观转录组学是一个不断扩大的研究领域，超过100种已知的RNA修饰。信使RNA （mRNA）上最常见的修饰是n6 -甲基腺苷（m6A）。虽然m6A在基因表达转录后调控中的功能开始被揭示，但m6A在复杂生物体发育过程中的确切作用仍不清楚。我们对黑腹果蝇的甲基转移酶复合体的各个组成部分以及YTH核解读蛋白进行了全面的分子和生理表征。我们证明了该复合物在神经元功能和性别决定中的重要作用，并暗示核读取器YT521-B在这些过程中是主要的m6A效应物。重要的是，我们确定了分裂端蛋白家族的成员，Spenito (Nito)，作为甲基转移酶复合物的一个新的真正的亚基。Nito与复合物的其他组分相互作用，其损失导致m6A水平的强烈下降。RMB15/RBM15B是Nito的脊椎动物同源基因，其在甲基转移酶复合体中的作用最近也得到了证实。它的缺失被证明会阻止复合体募集到XIST mRNA，导致m6A沉积失败，并促进x染色体沉默。因此，目前的模型提出RBM15/RBM15B通过其RNA结合域直接与mRNA相互作用，并通过促进甲基转移酶复合物募集到m6a靶向位点，从而提供特异性。然而，RBM15/RBM15B如何与甲基转移酶复合物的其他组分相互作用，以及其活性是否足以将复合物引导到RNA仍有待确定。在这个提议中，我们想要解开Nito在甲基转移酶复合体中的精确分子功能。结合生物化学和最先进的核糖组学方法，我们将系统地解决以下问题：(1)Nito与甲基转移酶复合体的其他成员之间的直接分子相互作用是什么？(2) m6A的功能及其生理活性需要Nito的哪些蛋白结构域？(3) Nito的直接靶点是什么？是否需要将甲基转移酶复合物引导到目标位点？(4) Nito是否足以招募甲基转移酶复合物并促进m6A从头生成。我们的研究将使我们能够详细了解甲基转移酶复合物内的Nito组装及其对m6A生物发生的精确贡献。

Epitranscriptomics is an expanding research area of more than 100 known RNA modifications. The most prevalent modification on messenger RNA (mRNA) is N6-methlyadenosine (m6A). While the functions of m6A in the posttranscriptional regulation of gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. We have carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex as well as of the YTH nuclear reader protein in Drosophila melanogaster. We demonstrated important roles of this complex in neuronal functions and sex determination, and implicate the nuclear reader YT521-B as a main m6A effector in these processes. Importantly, we identified the member of the split ends protein family, Spenito (Nito), as a novel bona fide subunit of the methyltransferase complex. Nito interacts with other components of the complex and its loss leads to strong decrease of m6A levels. RMB15/RBM15B is the vertebrate ortholog of Nito and its role within the methyltransferase complex was also recently demonstrated. Its absence was shown to prevent the recruitment of the complex to XIST mRNA, resulting in a failure to deposit m6A and to promote X-chromosome silencing. Hence, the current model proposes that RBM15/RBM15B interacts directly with mRNA via its RNA binding domains and in this way provides the specificity to the methyltransferase complex by promoting its recruitment to m6A-targeted sites. However, how RBM15/RBM15B interacts with other components of the methyltransferase complex and whether its activity alone is sufficient to guide the complex to RNA remains to be determined. In this proposal, we want to unravel the precise molecular function of Nito within the methyltransferase complex. Combining biochemistry and state-of-the-art ribonomics approaches, we will systematically address the following questions: (1) What are the direct molecular interactions between Nito and the other members of the methyltransferase complex? (2) Which protein domains of Nito are required for m6A functions and its physiological activities? (3) What are the direct Nito targets and is it required to guide the methyltransferase complex to its targeted sites? (4) Is Nito sufficient to recruit the methyltransferase complex and promote m6A de novo. Our study will enable us to obtain detailed insights into Nito assembly within the methlytransferase complex and its precise contribution to m6A biogenesis.