Department of Chemical and Systems Biology, Stanford University School of Medicine, 269 Campus Drive CCSR 4145 Stanford, CA 94305, United States.
Sarafan ChEM-H, Stanford University, Stanford ChEM-H Building 290 Jane Stanford Way Stanford, CA 94305, United States.
Glycobiology. 2024 Mar 19;34(1). doi: 10.1093/glycob/cwad076.
Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.
半乳糖凝集素-3（Galectin-3）作为一种糖结合蛋白而被广泛研究，现已被鉴定为一种假定的 RNA 结合蛋白，可能通过与剪接体的相互作用参与前体 mRNA 的成熟。鉴于细胞表面 RNA 生物学的最新发展，Galectin-3 的假定双重功能性质唤起了糖生物学和 RNA 生物学之间可能存在的非经典联系。然而，由于缺乏对直接 RNA 相互作用的功能证据，许多分子水平的观察依赖于亲和试剂，并且缺乏适当的遗传对照。因此，直接相互作用的证据仍然难以捉摸。我们证明，针对内源性人半乳糖凝集素-3 的抗体可以分离 RNA-蛋白交联，但这种活性仍然对 LGALS3 敲除不敏感。抗半乳糖凝集素-3 IP 的蛋白质组学分析表明，半乳糖凝集素-3 丰富，但富含 hnRNPA2B1，hnRNPA2B1 是一种丰富的、特征明确的 RNA 结合蛋白，与半乳糖凝集素-3 的 N 端结构域具有弱同源性，在分离物中高度富集。HNRNPA2B1 的遗传缺失，但不是 LGALS3 的缺失，消除了抗半乳糖凝集素-3 抗体分离 RNA-蛋白交联的能力，这意味着存在间接相互作用或交叉反应。为了解决这个问题，我们在 LGALS3 的内源性 C 端基因座引入了一个表位标签。未发现标记的半乳糖凝集素-3 与 RNA 发生任何蛋白交联。这一结果表明，半乳糖凝集素-3 与 RNA 没有直接相互作用，并且可能被错误地鉴定为 RNA 结合蛋白，至少在首次发现假定的 RNA 关联的 HeLa 细胞中是这样。我们鼓励进一步研究这一现象，采用基因缺失和在可能的情况下采用内源性表位标签，以达到评估潜在相互作用所需的特异性。
