Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.
Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, Ohio, USA.
J Biol Chem. 2024 Aug;300(8):107540. doi: 10.1016/j.jbc.2024.107540. Epub 2024 Jul 4.
Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal noncanonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of four putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in fragile X syndrome.
功能失调的脆性 X 智力低下蛋白（FMRP）的丧失会导致脆性 X 综合征，是自闭症谱系障碍和智力残疾的主要单基因病因。FMRP 主要是一种翻译抑制剂，据认为它通过使 FMRP 结合的多核糖体停滞来抑制翻译延伸，因为脑组织中的 FMRP 结合的多核糖体对嘌呤霉素和核酸酶处理具有抗性。在这里，我们提供的数据表明，FMRP 的 C 端非典型 RNA 结合域是诱导嘌呤霉素抗性 mRNA•核糖体复合物所必需和充分的。鉴于停滞的核糖体可以刺激核糖体碰撞和无意义 mRNA 衰变（NGD），我们测试了 FMRP 在神经母细胞瘤细胞中驱动其靶转录物 NGD 的能力。事实上，与对照相比，FMRP 和核糖体蛋白，但不是 poly(A)结合蛋白，在分离的核酸酶抗性双联体中富集。通过 siRNA 敲低和 RNA-seq，我们鉴定了 16 个可能由 FMRP 介导的 NGD 底物，其中许多编码参与神经元发育和功能的蛋白质。当 FMRP 耗尽或通过 RNAi 防止 NGD 时，四个假定底物的 mRNA 稳定性也增加。这些数据表明，FMRP 使核糖体停滞，但仅刺激一小部分特定转录物的 NGD，这揭示了 FMRP 的一个次要作用，在脆性 X 综合征中会失调。
