Table_2_HuD Binds to and Regulates Circular RNAs Derived From Neuronal Development- and Synaptic Plasticity-Associated Genes.XLSX

The RNA-binding protein (RBP) HuD is involved in neuronal differentiation, regeneration, synaptic plasticity and learning and memory. RBPs not only bind to mRNAs but also interact with several types of RNAs including circular RNAs (circRNAs), a class of non-coding RNAs generated by pre-mRNA back-splicing. This study explored whether HuD could regulate the expression of neuronal circRNAs. HuD controls target RNA’s fate by binding to Adenylate-Uridylate Rich Elements (AREs). Using bioinformatics analyses, we found HuD-binding ARE-motifs in about 26% of brain-expressed circRNAs. By RNA immunoprecipitation (RIP) from the mouse striatum followed by circRNA arrays, we identified over 600 circRNAs bound to HuD. Among these, 226 derived from genes where HuD also bound to their associated mRNAs including circHomer1a, which we previously characterized as a synaptic HuD target circRNA. Binding of HuD to two additional plasticity–associated circRNAs, circCreb1, and circUfp2, was validated by circRNA-specific qRT-PCR. Interestingly, we found that circUpf2 is also enriched in synaptosomes. Pathway analyses confirmed that the majority of HuD-bound circRNAs originate from genes regulating nervous system development and function. Using striatal tissues from HuD overexpressor (HuD-OE) and knock out (KO) mice for circRNA expression analyses we identified 86 HuD-regulated circRNAs. These derived from genes within the same biological pathways as the HuD RIP. Cross-correlation analyses of HuD-regulated and HuD-bound circRNAs identified 69 regulated in either HuD-OE or HuD-KO and 5 in both sets. These include circBrwd1, circFoxp1, and circMap1a, which derive from genes involved in neuronal development and regeneration, and circMagi1 and circLppr4, originating from genes controlling synapse formation and linked to psychiatric disorders. These circRNAs form competing endogenous RNA (ceRNA) networks including microRNAs and mRNAs. Among the HuD target circRNAs, circBrwd1 and circFoxp1 are regulated in an opposite manner to their respective mRNAs. The expressions of other development- and plasticity-associated HuD target circRNAs such as circSatb2, cirHomer1a and circNtrk3 are also altered after the establishment of cocaine conditioned place preference (CPP). Collectively, these data suggest that HuD interactions with circRNAs regulate their expression and transport, and that the ensuing changes in HuD-regulated ceRNA networks could control neuronal differentiation and synaptic plasticity.

RNA结合蛋白（RNA-binding protein, RBP）HuD参与神经元分化、再生、突触可塑性以及学习记忆过程。RBP不仅可结合mRNA，还能与多种类型的RNA相互作用，其中包括环状RNA（circular RNAs, circRNAs）——一类由前体mRNA反向剪接产生的非编码RNA。本研究探讨了HuD是否可调控神经元环状RNA的表达。HuD通过结合腺苷酸尿苷酸富集元件（Adenylate-Uridylate Rich Elements, AREs）来调控靶RNA的命运。通过生物信息学分析，我们在约26%的脑表达环状RNA中发现了HuD结合的ARE基序。利用小鼠纹状体的RNA免疫沉淀（RNA immunoprecipitation, RIP）结合环状RNA芯片技术，我们鉴定出600余种与HuD结合的环状RNA。其中226种环状RNA来源于HuD同时结合其相关mRNA的基因，包括我们此前已鉴定为突触HuD靶标环状RNA的circHomer1a。通过环状RNA特异性qRT-PCR，我们验证了HuD与另外两种与可塑性相关的环状RNA——circCreb1和circUfp2的结合。有趣的是，我们发现circUpf2同样在突触体中富集。通路分析证实，绝大多数与HuD结合的环状RNA来源于调控神经系统发育与功能的基因。我们使用HuD过表达（HuD-OE）小鼠和敲除（KO）小鼠的纹状体组织进行环状RNA表达分析，鉴定出86种受HuD调控的环状RNA。这些环状RNA来源于与HuD RIP靶基因处于同一生物学通路的基因。对受HuD调控的环状RNA与HuD结合的环状RNA进行交叉关联分析，发现69种环状RNA在HuD-OE或HuD-KO组中存在表达变化，另有5种在两组中均出现表达改变。其中包括来源于参与神经元发育与再生的基因的circBrwd1、circFoxp1和circMap1a，以及来源于调控突触形成且与精神疾病相关的基因的circMagi1和circLppr4。这些环状RNA可形成包含微小RNA（microRNAs, miRNAs）和mRNA的内源竞争RNA（competing endogenous RNA, ceRNA）网络。在HuD靶标环状RNA中，circBrwd1和circFoxp1的表达变化与它们各自对应的mRNA呈相反趋势。其他与发育和可塑性相关的HuD靶标环状RNA，如circSatb2、cirHomer1a以及circNtrk3的表达，在建立可卡因条件性位置偏好（cocaine conditioned place preference, CPP）模型后同样发生改变。综上，本研究数据表明，HuD与环状RNA的相互作用可调控其表达与运输，而HuD调控的内源竞争RNA网络的后续变化或可控制神经元分化与突触可塑性。