Project description:This study explores whether HuD could bind to and regulate the expression circRNAs from genes associated with neuronal development and synaptic plasticity circRNAs bound to HuD were isolated from the striatum of HuD-OE mice by RNA immunoprecipitation (RIP) as described in Bolognani et al, 2010 (https://doi.org/10.1093/nar/gkp863) using Dynabeads® (Thermo Fisher Scientific) coated with mouse monoclonal anti-myc tag antibody (9B11; Cell Signalling Technology Inc.) specifically recognizing myc-tagged HuD transgenic protein expressed in HuD-OE as described before (Bolognani et al., 2010; Zimmerman et al., 2020). Controls for RIP assays were performed using either non-immune IgG and HuD-OE tissue or the myc-tag antibody and wild type (WT) tissue.
Project description:Purpose: The goals of this study are to determine how deletion of the RNA-binding protein HuD affects the levels of microRNAs in the striatum of Elav4 (HuD) KO using miRNA-seq Methods: Total RNA from striatum of male adult Elavl4 (HuD) KO and wild-type (WT) littermates (n=3 per genotype) crossbred to C57Bl/6 for more than 10 generations were analyzed by small RNA sequencing, in triplicate, using an Illumina NextSeq platorm. The percentage of the number of bases with Q >30 for all 6 samples were >93%. Results: Using an optimized data analysis workflow (see diagram below), about 6.3 million sequence reads per HuD KO sample and 3.3 million per WT sample were mapped miRBase v22. The expression level (Reads count) of miRNAs were calculated using miRDeep2. The number of identified miRNA per group was calculated based on the mean of CPM in group > 1. Conclusions: We found 309 miRNAs from 54 unique families (FC>1.75- and p<0.05) were significantly upregulated while 161 miRNAs from 143 unique families (FC<0.55 and p<0.05) were significantly downregulated in the striatum of HuD KO mice vs WT samples.
Project description:Circular RNA (circRNA) has recently emerged as a novel type of endogenous non-coding RNA. It plays natural microRNA sponge effect that represses the activity of corresponding miRNAs by binding with them, thus regulating gene expression. Recent studies demonstrate that circRNAs are significantly enriched in the brain and a disproportionate fraction of them is derived from host genes that code for synaptic proteins. In addition, studies have shown that microRNAs are involved in synaptic plasticity, memory formation and cocaine addiction. However, the role of circRNAs in cocaine reward is largely unknown. This study aimed to identify the expression profile of circRNAs in the striatum of mice after cocaine self-administration by using circRNA microarray analysis.
Project description:Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. Besides binding and stabilizing the GAP-43 mRNA, HuD was shown to bind in vitro or in vivo the mRNAs of acetylcholinesterase, tau, p21, neuroserpin, and MARCKS among others. To identify additional targets of HuD, we propose to pull down the RNAs bound to myc-tagged HuD in vivo in the brains of HuD transgenic mice, to isolate these bound RNAs and use these to probe DNA microarrays. We will use pull downs using non-immune IgGs as controls. To test our hypothesis we propose 2 specific aims:; 1) To identify the targets of HuD in HuD overexpressor mice and; 2) To compare these target mRNAs to those we identified previously as having increased levels of expression in the hippocampus of HuD transgenic mice (see protocol # Perrone-Bizzozero-5R01NS030255-12) and/or those that show increased expression in dentate granule cells of HuD transgenic mice ( protocol # perro-affy-mouse-309741); Based upon the role of the RNA-binding HuD in neuronal development and synaptic plasticity, we expect that HuD targets will include mRNAs for proteins involved in these processes. All mice are in C57BL/6 background and are male approximately 60 days old. To identify target of HuD in our transgenic mice, we will homogenize the homogenize the hippocampi (2 per animal) of 3 transgenic mice and use these protein extracts for immunoprecipitation assays. Briefly, transgenic myc-tagged HuD protein will be immunoprecipitated using myc-tag antibodies and protein-G agarose beads and samples will be sent to T-Gen for RNA isolation, single round amplification and probing of DNA microarrays. We will use non-immune IgG as a negative control. Conditions:; Pooled Extracts from 3 transgenic mice IP with myc-tag antibodies: Triplicates; Pooled extract from 3 transgenic mice IP with non-immune IgGs (negative control): duplicates