Article Title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior
Figure Lengend Snippet: CLIP-seq analysis identified an increase in hnRNP H binding to the distal end of 3’UTR of Cacna2d2 in Hnrnph1 mutants that was associated with decreased usage of the 3’UTR, warranting further validation. (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 difference in usage of the proximal and distal end of the 3’UTR of Cacna2d2 as well as exons 37-38 and exons 3-4. The schematic indicates the positions of these primers. SAL = saline; MA = methamphetamine; WT = wild-types; H1 MUT = Hnrnph1 mutants. (B): The left striata were harvested from mice in the same way as in CLIP-seq followed by RNA extraction, cDNA library generation with oligo-DT primers, and RT-qPCR to detect differential usage of the regions at the 3’UTR of Cacna2d2 and upstream of it. No differences were found at exons 3-4 [F(1,39) Genotype x Treatment = 0.650, p = 0.425], exons 37-38 [F(1,39) Genotype x Treatment = 0.900, p = 0.349], or at the proximal end of the 3’UTR [F(1,39) Genotype x Treatment = 0.198, p = 0.659]. Although no significant Genotype x Treatment interaction was detected for the usage of the distal end of the 3’UTR [F(1,39) Genotype x Treatment = 1.485, p = 0.230], there was a significant main effect of Treatment [F(1,39) Treatment = 10.772, p = 0.002]. In examining the effect of Treatment at each level of Genotype, a significant, simple main effect of Treatment was found in the Hnrnph1 mutants [F(1,40) Treatment = 9.368, p = 0.004] but not in the wild-types [F(1,40) Treatment = 2.993, p = 0.091]. Subsequent analysis revealed decreased methamphetamine-induced usage of the distal end of 3’UTR in Hnrnph1 mutants compared to wild-types [t(40) = −3.061, *p = 0.004], with no significant genotypic difference between saline groups [t(40) = −1.730, p = 0.091]. The normalized 2 −ΔΔCT values from three independent replicates are shown in the plots. (C): The right striata of the same mice from the RT-qPCR analysis were collected for Western blot analysis to assess differences in CACNA2D2 protein expression. Immunoblots from three separate replicates are shown on the left with quantification shown on the right. There was no significant genotypic difference in the level of CACNA2D2 protein in saline or methamphetamine treatment groups [F(1,40) Genotype x Treatment = 2.587, p = 0.117]. (D): Schematics showing the putative interaction between hnRNP H-mediated selection of polyadenylation site in Cacna2d2. We proposed that binding of hnRNP H to the pA-3 site within the 3’UTR of Cacna2d2 blocks the usage of pA-3 to produce transcripts with a shorter 3’UTR in Hnrnph1 mutants in response to methamphetamine, (shown on right). This observation was identified in alternative splicing analysis ( , middle panel) and subsequently validated in the RT-qPCR ( , rightmost panel).
Article Snippet: Following the imaging, the membranes were blocked with 5% milk for 1 hand probed with anti-CACNA2D2 (1:1000; alomone, Cat#ACC-102) over night at 4°C followed by 1 hour probing with donkey anti-rabbit HRP (1:10,000; Jackson ImmunoResearch Labs Cat#711-035-152).
Techniques: Binding Assay, RNA Extraction, cDNA Library Assay, Quantitative RT-PCR, Western Blot, Expressing, Selection