HuD Binds to and Regulates Circular RNAs Derived From Neuronal Development- and Synaptic Plasticity-Associated Genes

Abstract

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.

The Biomarker and Therapeutic Potential of Circular Rnas in Schizophrenia

“…Recently, using bioinformatical prediction, it has been shown that neuronal HuD protein (ELAVL4) contained AU-rich elements that were attractive for hundreds of mice brain-expressed circRNAs. RNA immunoprecipitation confirmed this finding and showed that approximately 600 mice brain-expressed circRNAs could interact with HuD [112]; moreover, 226 of them originated from the previously described circHomer1a gene [28].…”

Interplay of RNA-Binding Proteins and microRNAs in Neurodegenerative Diseases

“…[129] is reduced in the frontal cortex of ALS patients [129] TAF15 miR-17-92 CDKN1A/p21 regulates miRNA biogenesis to decrease genes related to cell proliferation [132] is reduced in an SOD1 G93A mouse model [133] hnRNP A1 miR-590-3p n.d. is cooperatively regulated in neuronal maintenance ? [135] is increased in the blood cells of FTD patients [135] hnRNP H miR-663a CDK4, CDK6 is cooperatively regulated in cellular senescence by targeting genes related to the cell cycle [137] is increased in the blood cells of ALS patients [138] RALY miR-483 ATP5I, ATP5G1, ATP5G3, CYC1 regulates miRNA biogenesis to decrease genes related to mitochondrial metabolism [140] n.d. miR-676 miR-877 RBM17 miR-150-5p VEGF-A is cooperatively regulated in genes related to growth factors [143] is reduced in the CSF of ALS patients [145] HuD miR-125a-5p CAMSAP3 cooperatively regulates circRNA in aging by targeting genes related to neuronal polarization [148] is reduced in aging mice [147] Matrin-3 miR-138-5p n.d.…”

“…HuD has been reported to regulate ALS-associated SOD1 expression during oxidative stress in differentiated neuroblastoma cells and the sporadic ALS motor cortex [ 146 ]. HuD binds to and regulates circular RNAs derived from genes regulating neuronal development and synaptic plasticity and may act as a sponge for the miRNAs such as miR-125a-5p, which has previously been reported to be associated with aging [ 147 , 148 ]. Matrin-3 was recently found to be strongly linked to the pathogenesis of ALS [ 149 , 150 , 151 ] and has been shown to regulate processing of the synaptic miR-138-5p [ 152 ] ( Table 2 ).…”

RNA–Binding Protein HuD as a Versatile Factor in Neuronal and Non–Neuronal Systems

“…HuD–associated mRNAs have been analyzed by ribonucleoprotein immunoprecipitation (RIP) followed by microarray in the brains of transgenic mice [ 58 ], and also by CRAC (crosslinking and analysis of cDNA) in motor neuron cells expressing His–HA–HuD [ 52 ]. Recently, a series of HuD–associating circular RNAs (circRNAs) from HuD transgenic mice were identified by RIP analysis followed by circRNA arrays [ 60 ]. These analyses revealed that HuD interacts with a variety of mRNAs as well as non–coding RNAs, via their ARE regions and provided useful information concerning the roles of HuD in RNA regulation.…”

“…In addition to targets found in neuronal tissues, HuD promotes stabilization of target mRNAs also expressed in other tissues, such as p21 [ 83 ], Ca 2+ /Calmodulin–dependent protein kinase II α ( CaMKII α) [ 84 ], and musashi 1 ( MSI1 ) [ 85 ]. Additionally, an interesting study recently demonstrated that circRNAs, such as cirHomer1a , could be molecular targets of HuD [ 60 , 86 ].…”