Zhang Shengping, Zhang Yunlong, Chen Ting, Hu Hong-Yu, Lu Changrui
College of Biological Science and Medical Engineering, Donghua University, Shanghai 201620, China.
Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
Cells. 2025 Mar 6;14(5):383. doi: 10.3390/cells14050383.
Ataxin-2 (Atx2), an RNA-binding protein, plays a pivotal role in the regulation of RNA, intracellular metabolism, and translation within the cellular environment. Although both the Sm-like (LSm) and LSm-associated (LSmAD) domains are considered to associated with RNA binding, there is still a lack of experimental evidence supporting their functions. To address this, we designed and constructed several recombinants containing the RNA-binding domain (RBD) of Atx2. By employing biophysical and biochemical techniques, such as EMSA and SHAPE chemical detection, we identified that LSm is responsible for RNA binding, whereas LSmAD alone does not bind RNA. NMR and small-angle X-ray scattering (SAXS) analyses have revealed that the LSmAD domain exhibits limited structural integrity and poor folding capability. The EMSA data confirmed that both LSm and LSm-LSmAD bind RNA, whereas LSmAD alone cannot, suggesting that LSmAD may serve as an auxiliary role to the LSm domain. SHAPE chemical probing further demonstrates that LSm binds to the AU-rich, GU-rich, or CU-rich sequence, but not to the CA-rich sequence. These findings indicate that Atx2 can interact with the U-rich sequences in the 3'-UTR, implicating its role in poly(A) tailing and the regulation of mRNA translation and degradation.
ataxin-2（Atx2）是一种RNA结合蛋白，在细胞环境中RNA的调控、细胞内代谢及翻译过程中发挥着关键作用。尽管类Sm（LSm）结构域和LSm相关（LSmAD）结构域都被认为与RNA结合有关，但仍缺乏支持其功能的实验证据。为解决这一问题，我们设计并构建了几种包含Atx2 RNA结合结构域（RBD）的重组体。通过采用生物物理和生化技术，如电泳迁移率变动分析（EMSA）和选择性2'-羟基酰化引物延伸（SHAPE）化学检测，我们确定LSm负责RNA结合，而单独的LSmAD不结合RNA。核磁共振（NMR）和小角X射线散射（SAXS）分析表明，LSmAD结构域的结构完整性有限且折叠能力较差。EMSA数据证实LSm和LSm-LSmAD都能结合RNA，而单独的LSmAD则不能，这表明LSmAD可能对LSm结构域起辅助作用。SHAPE化学探针进一步证明，LSm能结合富含AU、GU或CU的序列，但不结合富含CA的序列。这些发现表明，Atx2可与3'-非翻译区（UTR）中富含U的序列相互作用，这暗示了其在多聚腺苷酸化以及mRNA翻译和降解调控中的作用。
