Neuroscience Graduate Program, Brown University, Providence, RI 02912, USA.
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912, USA.
Nucleic Acids Res. 2020 Oct 9;48(18):10542-10554. doi: 10.1093/nar/gkaa710.
hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.
hnRNPA2 是少突胶质细胞和神经元中 mRNA 运输颗粒的主要成分。然而，hnRNPA2 如何结合 A2 识别元件（A2RE）的结构细节，以及该序列是否通过增强 hnRNPA2 的相分离来刺激颗粒形成，尚未得到研究。通过溶液 NMR 和生物物理研究，我们发现两个单独的 RRM 中的每一个都保留了与 RNA 复合物中观察到的结构域结构，但在没有 RNA 的情况下，它们在溶液中并不刚性限制（即它们独立移动）。hnRNPA2 RRM 弱但至少可以结合最小的 rA2RE11，并且很可能两个 hnRNPA2 分子能够同时结合更长的 21 mer 髓鞘碱性蛋白 A2RE。在 RNA 结合后，两个 RRM 中都观察到 NMR 化学位移偏差，表明它们都在结合 A2RE11 中起作用。有趣的是，添加短 A2RE RNA 或包含该序列的更长 RNA 完全阻止全长 hnRNPA2 的体外相分离和疾病相关突变体的聚集。这些发现表明，RRM 与特定识别序列的相互作用本身并不能解释颗粒形成的成核作用，这与模型一致，即在颗粒蛋白和长 RNA 转录本中，多个蛋白质：RNA 和蛋白质：蛋白质接触形成跨越许多位点。
