The striatal heterogeneous nuclear ribonucleoprotein H mRNA targetome associated with methamphetamine administration and behavior

Figures

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  Figure 1.. Striatal RNA-binding sites of hnRNP H at baseline (saline control) are enriched for introns and G-rich binding motifs. eCLIP-seq revealed transcriptome-wide striatal RNA targets of hnRNP H in saline control wild-types (WT_SAL). Peak calling of both uniquely and multi-mapped reads was performed using CLAM (Zhang and Xing, 2017). (A): RNA 32P autoradiogram of hnRNP H-bound RNA. CLIP conditions are shown for each lane: no crosslinking, IgG mock IP, and four different RNase If concentrations from high to low. As expected, an increasing amount of RNA was pulled down as the concentration of RNase If was decreased. The scissors denote the region above the molecular weight of hnRNP H (50 kDa) that was isolated for sequencing. The region runs from 50 to 80 kDa, which corresponds to 30 to 70 bp for RNA length. The larger bands that were greater than 100 kDa could represent very long RNAs that are resistant to digestion or large hnRNP H-associated protein-protein complexes bound to RNAs. (B): hnRNP H binds primarily to introns of target RNAs. More than 70% of hnRNP H CLIP sites are intronic. The pie chart shows the relative distribution of CLIP sites in 5’UTR, coding sequences (CDS),…

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  Figure 2.. Methamphetamine-induced striatal RNA-binding targets of hnRNP H are enriched for pathways and cellular components involved in drug-evoked synaptic plasticity. CLIP-seq analysis revealed transcriptome-wide striatal RNA-binding targets of hnRNP H at 30 min post-saline (i.p.) versus methamphetamine treatment (2 mg/kg, i.p.). Peak calling in CLAM (Zhang and Xing, 2017) was performed separately for both conditions. For differential analysis, peak calling was also performed using CLAM (Zhang and Xing, 2017) on the merged bam file across the two conditions followed by read counts against the identified peaks and differential analysis of peak intensity. SAL = saline; MA = methamphetamine. (A): In comparison to SAL, only the percentages of hnRNP H binding events associated with 3’UTR and introns were significantly different in the other three conditions (See chi-square tests in Table S5). The percentage of binding events that comprised 3’UTRs varied robustly between SAL vs MA (chi-square test: *p < 0.001). All intron binding events (including distal introns and proximal introns) were significantly different from SAL (chi-square test: all p’s < 0.001), The relative distribution of…

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  Figure 3.. 3’UTR targets of hnRNP H show a methamphetamine-induced increase in binding in striatum . Methamphetamine-induced hnRNP H CLIP peaks, stratified across four gene elements, including 5’UTR, 3’UTR, intron, and CDS. SAL = saline; MA = methamphetamine. Heatmaps show normalized log2 CPM (count-per-million) read counts for significant peaks showing a MA treatment effect. The row of each heatmap represents a binding site sorted by p-value from smallest to largest, with those with a p-value of less than 0.05 shown in the heatmap. Each column represents a sample, with the first three samples (grouped by the grey bar) being SAL-treated and the final 3 samples (grouped by the pink bar) being MA-treated. In response to MA, there was an increase in baseline binding of hnRNP H to the 3’UTR regions of RNA-binding targets.

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  Figure 4.. RNA targets showing a methamphetamine-induced change in hnRNP H1 binding, gene expression, and alternative splicing in striatum. Differential gene expression (DE) comparing MA vs SAL was performed using DESeq2 (Love et al., 2014). Differential exon and intron usage analysis (alternatively spliced; AS) for the effect of methamphetamine was performed using Aspli (Mancini et al., 2020). SAL = saline; MA = methamphetamine. (A): Venn diagram comparing RNA-binding targets of hnRNP H with differential gene expression and differential exon or intron usage. The 64 DE genes and 260 AS genes were compared with the 327 hnRNP H targets that showed differential binding in response to methamphetamine. The overlapping genes are highlighted in red. (B): Volcano plot of differentially expressed genes in response to methamphetamine.

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  Figure 5.. Methamphetamine-induced change in hnRNP H1 binding, gene expression, and alternative splicing of Cacana2d2 in striatum. (A): hnRNP H preferentially binds to the 3’UTR of Cacna2d2. Visualization of reads by Integrative Genome Browser (Thorvaldsdóttir et al., 2013) for the hnRNP H CLIP peak at the 3’UTR of Cacna2d2. Scale of the plot height is in counts per million (CPM). (B): A G-rich motif was detected at the hnRNP H CLIP peak within the 3’UTR of Cacna2d2. De novo motif discovery of the binding site was performed in MEME (Bailey et al., 2009). (C): Effect plots showing methamphetamine-induced decrease in hnRNP H binding to the 3’ UTR of Cacna2d2 [left; t(4) = 2.346, p = 0.0789], increased usage of the 3’UTR [middle, t(4) = 2.315, p = 0.0816], and a statistically significant decrease in transcript levels of Cacna2d2 (right, [t(4) = 3.317, p = 0.0295). Here, 3’UTR usage is defined as to the number of normalized reads mapped to the 3’UTR portion of Cacna2d2. The effect plots were generated from CLIP-seq and RNA-seq data showing average values with standard error of the means, with n = 3 per condition.

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  Figure 6.. The CACNA2D2/CACNA2D1 inhibitor pregabalin selectively reduces methamphetamine-induced locomotor activity. On Days 1 and 2 (habituation days), 30 min after pretreatment with saline (i.p.) and placement back into the home cage, mice were injected again with saline (i.p.) and placed into testing apparatus for 1 h. On Day 3, approximately one-half of the mice were pretreated first with saline (i.p.) while the other half received pregabalin (30 mg/kg, i.p.) and were placed back into the home cage for 30 min. Mice were then injected with 2 mg/kg methamphetamine (i.p.) or saline (i.p.) and immediately placed into the testing apparatus for 60 min followed by immediate sacrifice and removal of the striatum. SAL_SAL = saline pre-treatment followed by saline; Pregabalin_SAL = pregabalin pre-treatment followed by saline; SAL_MA = SAL pre-treatment followed by MA; Pregabalin_MA = pregabalin pre-treatment followed by. (A): Injection timeline for those mice pre-treated with pregabalin (30 mg/kg, i.p.) and methamphetamine (2 mg/kg, i.p.) on Day 3. (B-C): Locomotor activity on Day 1 (B) and day 2 (C) for 1 hr in 5-min bins. Mice assigned for eventual pretreatment with saline or pregabalin…

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  Figure 7.. Pregabalin pretreatment increases proximal 3’ UTR usage of Cacna2d2 in striatum. 60 min post-saline or methamphetamine injection (90 min post-saline or pregabalin pretreatment), mice from the locomotor study (Figure 6) were sacrificed and whole striatum for each mouse was dissected and harvested for RT-qPCR to detect differential 3’UTR usage of Cacna2d2. SAL = saline; MA = methamphetamine; PGB = pregabalin. (A): Three polyadenylation sites (pA-1, pA-2, and pA-3) are present within the 3’UTR of Cacna2d2 that distinguish isoforms containing 3’UTR of different lengths (UCSC Genome Browser). Primers were designed to detect differences in usage of the proximal and distal end of the 3’UTR of Cacna2d2 (UCSB annotations). The schematic indicates the positions of these primers. (B-D): In examining the effect of pregabalin pretreatment (30 mg/kg, i.p.) followed by saline (i.p.), there was no difference in the usage of the proximal 3’UTR of Cacna2d2 (B) [t(22) < 1, p = 0.811], no difference in the usage of the distal 3’UTR of Cacna2d2 (C) [t(22) = 1.791, p = 0.087], and no difference in Cacna2d1 transcript levels (D) [t(22) < 1, p = 0.819]. (E-G): Pre-treatment with pregabalin (30 mg/kg,…