ECT2 peptide sequences outside the YTH domain regulate its m⁶A-RNA binding

The m⁶A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m⁶A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In <i>Arabidopsis thaliana</i> IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved <i>C</i>-Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an <i>in vitro</i> biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m⁶A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m⁶A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m⁶A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA <i>in vivo</i>. Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m⁶A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

N⁶-甲基腺嘌呤（m⁶A）表观转录组修饰是目前已知丰度最高、分布最广泛的RNA内部化学修饰。该修饰通过调控RNA代谢过程，成为真核生物生殖、生长、形态发生以及应激响应的关键调控因子。核心的m⁶A阅读蛋白构成了一个拥有数百个成员的蛋白质超家族，这类蛋白均携带保守的YTH RNA结合结构域（YTH RNA binding domain）。绝大多数YTH家族蛋白仅含有固有无序区域（Intrinsically Disordered Region, IDR），并无其他显著额外结构域。在拟南芥（Arabidopsis thaliana）中，固有无序区域对不同YTH蛋白的功能特化至关重要，这类蛋白被命名为进化保守C端区域（Evolutionarily Conserved C-Terminal region, ECT）1至12。本研究以拟南芥ECT2蛋白为研究对象，通过体外（in vitro）生化实验表征，证实全长ECT2蛋白及其单独的YTH结构域均具备结合m⁶A的独特能力，这与此前已报道的YTH家族阅读蛋白特性有所不同。我们鉴定出ECT2的YTH结构域之外的肽段区域——位于其N端固有无序区域内——可调控其与m⁶A甲基化RNA的结合能力。进一步研究发现，ECT2结合m⁶A的选择性可被其邻近序列的高尿苷含量所增强，而此前研究认为ECT2的N端固有无序区域可在体内（in vivo）与靶标RNA直接结合。此外，我们还鉴定出一类位于ECT2 YTH结构域旁侧、在大量YTH家族蛋白中保守存在的小型结构元件，该元件可增强其与m⁶A甲基化RNA的结合能力。基于上述研究结果，我们提出：这类调控区域不仅存在于开花植物的ECT家族或YTH阅读蛋白中，也可能广泛分布于真核生物的各类YTH家族阅读蛋白之中。