Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay
Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay
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DOI：
10.1016/j.bbrc.2023.03.047
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发表时间：
2023-03-26
影响因子：
3.1
通讯作者：
Hayashi，Ken'ichiro
中科院分区：
文献类型：
--
作者：
Morita，Tsuyoshi;Hayashi，Ken'ichiro
肌动蛋白相关蛋白5（ARP 5）抑制骨骼肌、平滑肌和心肌组织的分化，并且ARP 5表达根据肌肉分化状态的生理和病理变化而增加或减少。然而，ARP 5表达的调控机制在很大程度上是未知的。在这里，我们确定了一个novelArp 5 mRNA的异构体，包含提前终止密码子的选择外显子7 b，因此无义介导的mRNA衰变（NMD）的目标。在小鼠骨骼肌细胞中，从经典Arp 5亚型，即，Arp 5（7a）与NMD靶向亚型Arp 5（7 b）的转化发生在分化过程中，表明Arp 5的表达受NMD偶联的选择性剪接（AS-NMD）的调控。我们开发了一种新颖的方法来准确定量两种Arp 5亚型的比例，并测量了肌肉和脑组织中较高水平的Arp 5（7 b），其中Arp 5表达较少。Arp 5外显子7的3′剪接位点有一个不寻常的受体序列，这常常导致真正的剪接位点被跳过，并使用位于下游16个碱基的隐蔽剪接位点。当不寻常的受体序列突变为通常的受体序列时，Arp 5（7 b）亚型几乎检测不到。肌肉分化后，参与3′剪接位点识别的几种剪接因子的表达降低。此外，剪接因子的敲低增加了Arp 5的水平（7 b），降低了Arp 5的表达（7a）。此外，在人骨骼肌和心肌组织中发现Arp 5表达与这些剪接因子的水平之间存在强正相关。因此，Arp 5在肌肉组织中的表达很可能是由AS-NMD途径调节的。
Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novelArp5mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonicalArp5isoform, i.e.,Arp5(7a), to the NMD-targeted isoformArp5(7b)occurred during differentiation, suggesting thatArp5expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of bothArp5isoforms and measured higher levels ofArp5(7b)in muscle and brain tissues, where ARP5 is less expressed. The 3′ splice site inArp5exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, theArp5(7b)isoform was barely detectable. The expression of several splicing factors involved in 3′ splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels ofArp5(7b)and decreased the expression ofArp5(7a). Furthermore, strong positive correlations were found betweenArp5expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus,Arp5expression in muscle tissues is most likely regulated by the AS-NMD pathway.