Journal: bioRxiv

Article Title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

doi: 10.1101/2021.07.06.451358

Figure Lengend Snippet: Differential gene expression (DE) for the interaction of Genotype and Treatment [(MUT_MA – MUT_SAL) – (WT_MA – WT_SAL)] was performed using limma and edgeR . Differential exon and intron usage analysis (alternatively spliced; AS) for interaction of Genotype and Treatment [(MUT_MA – MUT_SAL) – (WT_MA – WT_SAL)] was performed using Aspli . (A): Venn diagram comparing RNA-binding targets of hnRNP H with differential gene expression and differential exon or intron usage. The 19 DE genes and 469 AS genes were then compared with the hnRNP H targets that comprised the Genotype x Treatment interactions. Cacna2d2 is the only RNA-binding target of hnRNP H that was both a DE gene and an AS gene. Only the top overlapping and non-overlapping hnRNP H targets and AS genes are indicated. Out of the 19 DE genes, Cacna2d2, Elfn1 , Calb2 , and Zic1 are putative targets of Malat1 (predicted by TargetScan; ) while Camta1 and Pcdh8 are putative targets of Mir124a (predicted by LncRRIsearch; ). (B): Volcano plot of genes showing a Genotype x Treatment interaction in response to methamphetamine. The interaction is expressed as (MUT_MA – MUT_SAL) – (WT_MA – WT_SAL). Cacna2d2 is circled in red. The five overlapping hnRNP H targets and DE genes are circled in purple. (C): hnRNP H preferentially binds to the 3’UTR of Cacna2d2 . Visualization of reads by Integrative Genome Browser for the hnRNP CLIP peak at the 3’UTR of Cacna2d2 . Scale of the plot height is in counts per million (CPM). (D): 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 . (E): Interaction plots showing hnRNP H binding to the 3’UTR of Cacna2d2 (left), differential usage of the 3’UTR (middle), and differential gene expression of Cacna2d2 (right) as a function of Genotype and Treatment. The increase in hnRNP H binding to the 3’UTR of Cacna2d2 was associated with decreased 3’UTR usage and increased gene expression of Cacna2d2 in Hnrnph1 mutants. Here, 3’UTR usage is defined as to the number of normalized reads mapped to the 3’UTR portion of Cacna2d2 . The interaction plots were generated from CLIP-seq and RNA-seq data showing average values with standard deviation of the means, with n = 3 per condition.

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: Expressing, RNA Binding Assay, Binding Assay, Generated, RNA Sequencing Assay, Standard Deviation

Journal: bioRxiv

Article Title: The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

doi: 10.1101/2021.07.06.451358

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

Journal: Stem Cell Research & Therapy

Article Title: Global DNA methylation pattern involved in the modulation of differentiation potential of adipogenic and myogenic precursors in skeletal muscle of pigs

doi: 10.1186/s13287-020-02053-3

Figure Lengend Snippet: CACNA2D2 negatively regulated myogenic differentiation by inhibiting JNK/MAPK signaling pathway. a Immunofluorescent microscopy analysis of the morphological changes and expression of Myosin in myogenic precursors transfected with CACNA2D2-plasmid or empty vector at 48 h of differentiation. Scale bars, 100 μm. b The mRNA expression level of CACNA2D2 and c – f protein level of CACNA2D2, MyoG, JNK/MAPK signaling key factors (JNK and c-Jun), and their phosphorylated forms (p-JNK and p-c-Jun) were measured in myogenic precursors transfected with CACNA2D2-plasmid or empty vector in growth medium (GM, 0 h) or after 12, 24, and 48 h switched into differentiation medium (DM). GAPDH was used as the internal control. Data were presented as means ± SEM ( n = 3). The statistical significance of difference between two means was calculated using t test, ** P < 0.01

Article Snippet: Antibody for CACNA2D2 (CSB-PA889145ESR2HU) was obtained from Cusabio ( http://www.cusabio.cn/ ).

Techniques: Microscopy, Expressing, Transfection, Plasmid Preparation

Journal: American Journal of Translational Research

Article Title: miR-1231 exacerbates arrhythmia by targeting calciumchannel gene CACNA2D2 in myocardial infarction

doi:

Figure Lengend Snippet: Putative target genes for miR-1231

Article Snippet: After that, PVDF membrane was incubated overnight at 4°C with primary antibodies forβ-Actin (Santa Cruz, SC-47778) and cacna2d2 (Novus Biologicals, NBP1-81501).

Techniques:

Journal: American Journal of Translational Research

Article Title: miR-1231 exacerbates arrhythmia by targeting calciumchannel gene CACNA2D2 in myocardial infarction

doi:

Figure Lengend Snippet: miR-1231 suppresses cacna2d2 expression by targeting its mRNA. (A) The mRNA level of cacna2d2 was significantly decreased in both human and rat ischemic heart samples. Real-time PCR analysis of the expression of predicted 14 ion channel target genes in ischemic hearts compared with the healthy control heart samples in both human and rats as depicted in Figure 1. In human samples, the randomly selected three pairs of human control non-ischemic hearts and MI hearts were compared, and in rat samples, three randomly chosen pairs of NIZ, BZ, IZ and Ctl samples from rats at 14 days post-MI were compared as well. Results of MI or IZ relative to Ctl were shown (*, P<0.05 compared with Ctl group, n=3). (B) Schematic illustration of sequence complimentarity between miR-1231 and the 3’-UTRs of cacna2d2 mRNAs in human (hsa) and rat (rno), provided with computational and bioinformatics-based approach using TargetScan [2]. Watson-Crick complementarity was presented in bold text and linked with its paired nucleotide. (C) 3’-UTR of human or rat cacna2d2 was target for human or rat miR-1231 in HEK293 cells, respectively. HEK293 Cells were transiently transfected with luciferase reporters linked to the 3’-UTR sequences of human or rat CACNA2D2 gene as indicated. The luciferase reporter activity was measured after 1 day co-expression. Human or rat miR-1231 repressed luciferase reporter gene activity, however, mutatedmiR-1231 was unable to decrease luciferase activity. Mean ± SD; n=8; *P<0.05; NS, not significant compared with Ctl). Unpaired Student’s t-test. (D, E) The mRNA and protein expression of cacna2d2 were downregulated by miR-1231 in cultured myocytes. The protein (D) and mRNA (E) levels of cacna2d2 were determined by Western blotting and qRT-PCR in cultured neonatal rat cardiac myocytes, which were transfected with miR-1231 alone or in combination with anti-miR-1231or anti-mutated. Left, β-Actin was used as a loading control and representative results of Western blotting bands were shown; Right, mRNA levels in four groups relative to Ctl were shown (*, P<0.05; NS, not significant compared with Ctl group, n=3).

Article Snippet: After that, PVDF membrane was incubated overnight at 4°C with primary antibodies forβ-Actin (Santa Cruz, SC-47778) and cacna2d2 (Novus Biologicals, NBP1-81501).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Sequencing, Transfection, Luciferase, Activity Assay, Cell Culture, Western Blot, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: miR-1231 exacerbates arrhythmia by targeting calciumchannel gene CACNA2D2 in myocardial infarction

doi:

Figure Lengend Snippet: miR-1231 suppresses cacna2d2 expression in ischemic hearts. A. The protein levels of cacna2d2 were reduced in both human and rat ischemic hearts. As depicted in Figure 1A, three pairs of human IZ and Ctl samples or rat NIZ, BZ, IZ and Ctl samples were subjected to Western blotting assay to detect cacna2d2 protein expression. β-Actin was used as a loading control. Top, representative results of Western blotting bands; bottom, quantitation relative to Ctl as mean ± SD (n=3). (*, P<0.05; NS, not significant compared with Ctl group). Unpaired Student’s t-test. B. The protein level of cacna2d2 in IZ zone recovered upon in vivo miR-1231 knockdown. Relative protein levels of cacna2d2 in rat hearts after in vivo transfer of saline, anti-miR-1231 or anti-mutated were measured by Western blotting analysis. β-Actin was used as a loading control. Top, representative results of Western blotting bands; bottom, quantitation relative to Ctl as mean ± SD (n=3). (*P<0.05; NS, not significant compared with Ctl). Unpaired Student’s t-test. C. miR-1231 levels in IZ zone decreased in response to in vivo knockdown. Relative levels of miR-1231 in rat hearts after in vivo transfer of saline, anti-miR-1231 or anti-mutated were measured by real-time PCR analysis. All values were normalized to non-ischemic hearts (Ctl, n=4). (*P<0.05; NS, not significant compared with group receiving no injection (None). Unpaired Student’s t-test. D. The protein level of cacna2d2 in IZ zone decreased after in vivo enforced expression of miR-1231. Relative protein level of cacna2d2 in rat hearts after in vivo transfer of adenovirus control or different amount of adenovirus-miR-1231 as indicated were analyzed by Western blotting. β-Actin was used as a loading control. Left, representative results of Western blotting bands; right, quantitation relative to adenovirus Ctl as mean ± SD (n=3). (*P<0.05; NS, not significant compared with adenovirus control). Unpaired Student’s t-test. E. miR-1231 levels in IZ zone increased after in vivo enforced expression. Relative levels of miR-1231 in rat hearts after in vivo transfer of adenovirus control or different amount of adenovirus-miR-1231 were measured by real-time PCR analysis. All values were normalized to adenovirus control (Ctrl, n=4). *P<0.05 compared with adenovirus control. NS, not significant compared with adenovirus control. Unpaired Student’s t-test.

Article Snippet: After that, PVDF membrane was incubated overnight at 4°C with primary antibodies forβ-Actin (Santa Cruz, SC-47778) and cacna2d2 (Novus Biologicals, NBP1-81501).

Techniques: Expressing, Western Blot, Quantitation Assay, In Vivo, Real-time Polymerase Chain Reaction, Injection

Journal: American Journal of Translational Research

Article Title: miR-1231 exacerbates arrhythmia by targeting calciumchannel gene CACNA2D2 in myocardial infarction

doi:

Figure Lengend Snippet: Silence of cacna2d2 induces arrhythmias in ischemic hearts. (A, B) Knockdown of cacna2d2 by siRNA induces arrhythmias in ischemic hearts of rats. Both the incidence of VT (A) and VF (B) in ischemic hearts increased following siRNA-mediated cacna2d2 silencing in the presence or absence of anti-miR-1231 treatment. Scrambled siRNA was used as a negative control (siCtrl). Data are presented as percent incidence (mean ± s.e.m.) in rat hearts at 14 days post-MI. Three independent experiments were conducted and each group contained 8 rats. *P<0.05; NS, not significant compared with control group without siRNA administration χ2-test. (C) The protein level of cacna2d2 in IZ zone was dramatically downregulated upon siRNA targeting. The protein level of cacna2d2 was measured by Western blotting analysis. β-Actin was used as a loading control and representative results of Western blotting bands were shown. (D) The level of miR-1231 was effectively reduced when anti-miR-1231 was utilized. Values were normalized to ischemic hearts receiving no treatment of anti-miR-1231. *, P<0.05. Unpaired Student’s t-test.

Article Snippet: After that, PVDF membrane was incubated overnight at 4°C with primary antibodies forβ-Actin (Santa Cruz, SC-47778) and cacna2d2 (Novus Biologicals, NBP1-81501).

Techniques: Negative Control, Western Blot

Journal: Frontiers in Microbiology

Article Title: Screening and Identification of Lujo Virus Entry Inhibitors From an Food and Drug Administration-Approved Drugs Library

doi: 10.3389/fmicb.2021.793519

Figure Lengend Snippet: Knockdown of voltage-gated calcium channel (VGCC) genes inhibited LUJVpv infection. (A) Western blotting (WB) assay of CACNA1S and CACNA2D2 in U-2 OS cells. U-2 OS cells were transfected with si CACNA1S and si CACNA2D2 , respectively. After 48 h, cells were infected with LUJVpv (MOI: 0.1) for 6 h. Then, the cell lysates were subjected to a WB assay. (B) Quantification results of WB assay. (C,D) Knockdown of VGCC genes inhibited LUJVpv infection. U-2 OS cells were transfected with si CACNA1S or si CACNA2D2 . After 48 h, cells were infected with LUJVpv (MOI: 0.1), MACVpv (MOI: 0.1), or VSVpv (MOI: 0.1). After 6 h, the duplicate cell lysates were subjected to qPCR (C) and Rluc assays (D) , respectively. Data are presented as presented as means ± SD from more than five independent experiments ( **** p < 0.0001; *** p < 0.001; ** p < 0.01; and * p < 0.05).

Article Snippet: The antibodies used in the Western blotting (WB) assay included anti-CaV1.1 mAB (Invitrogen; 1:1,000), anti-CACNA2D2 antibody (Abcam, Cambridge HQ, United Kingdom; 1:1,000), anti-β-tubulin mouse mAb (AC021; ABclonal, Wuhan, China; 1:1,000), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG, and HRP-linked goat anti-mouse IgG (1:5,000; Proteintech).

Techniques: Infection, Western Blot, Transfection