Journal: bioRxiv

Article Title: Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

doi: 10.1101/244905

Figure Lengend Snippet: (A) GO functional annotation clustering analysis for the genes used in . Red bars represent the GO term having p<0.05. (B) Comparative granule cell layer thickness. DAPI staining images in 4 weeks and 12 months old Control tdTomato; Pcp2-Cre and Nova2-cKO tdTomato; Pcp2-Cre and Calbindin D28 immunostaining images at 12 months old. (C) The climbing fiber innervation defect and reduced Purkinje molecular layer thickness upon Purkinje cell specific Nova2 deficiency. vGlut2 ( green , climbing fiber terminal marker), calbindin D28 ( red , Purkinje cells marker), and NeuN ( blue , granule cell marker) immunofluorescent staining images in the 4 weeks old Control Pcp2-Cre and Nova2- cKO Pcp2-Cre . (D) The swelling Purkinje cell’s neurites upon Nova2 deficiency. ITPR1 ( green ), Caibindin D28 ( red ), and NeuN ( blue ) immunofluorescent staining images from 16 weeks old Nova2 -cKO Pcp2-Cre mouse. Purple and blue arrowheads represent swelling neurites in molecular layer or granule cell layer, respectively.

Article Snippet: Primary antibodies used for CLIP, immunofluorescent staining (IF), and western blots (WB) were: mouse anti-GFP ( , clones 19F7 and 19C8, cTag-CLIP), rat anti-GFP (nacalai tesque, GF090R, IF: 1/1,000), mouse anti-GFP (Santa Cruz, sc-9996, WB: 1/1,1000), rabbit anti-mRFP/ tdTomato (ROCKLAND, 600–401–379, IF: 1/2,000), rabbit anti-NeuN/RBFOX3 (Millipore, ABN78, IF: 1/1,000), guinea pig anti-NeuN/RBFOX3 (Millipore, ABN90P, IF: 1/1,000), goat anti-NOVA2 (Santa Cruz, sc-10546, IF; 1/500, WB; 1/2,000, CLIP), rabbit anti-NOVA1 [EPR13847] (abcam, ab183024, IF: 1/1,1000), human anti-pan NOVA (anti-NOVA paraneoplastic human serum, WB: 1/5,000), rabbit anti-PTBP2 ( , IF:1/5,000, CLIP), guinea pig anti-vGlut1 (Synaptic System, 135 304, IF:1/2,000), rabbit anti-vGlut2 (Synaptic System, 135 403, IF:1/2,000), mouse anti-VGAT (Synaptic System, 131 011, IF: 1/1,000), rabbit anti-Calbindin D28 (Millipore, AB1778, IF: 1/1,000), goat anti-Calbindin D28 (Santa Cruz, sc-7691, IF: 1/500), rabbit anti-ITPR1/InsP3R, type1 (Millipore, ABS55, IF:1/2,000), mouse anti-GAPDH (Santa Cruz, sc-32233, WB: 1/1,000).

Techniques: Functional Assay, Staining, Immunostaining, Marker

Journal: bioRxiv

Article Title: Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

doi: 10.1101/244905

Figure Lengend Snippet: (A) Selective NOVA2 depletion from PCs. NOVA2 ( green ), calbindin D28 (calb, red: PCs marker), and NeuN ( blue: GCs marker) immunofluorescent staining images in the 4 weeks old cerebellum of Control Pcp2-Cre and Nova2-cKO Pcp2-Cre . (B) Progressive cerebellar atrophy in the PCs specific Nova2 -cKO mouse. Brain images of 16 weeks old Control Pcp2-Cre and Nova2 -cKO Pcp2-Cre mouse ( left ). Tissue weight of cerebellum and the other brain regions in 4 weeks old (4W) or 16 weeks old (16W) Control Pcp2-Cre and Nova2 -cKO Pcp2-Cre mouse ( right ) (n=3, *; p<0.05). (C) Progressive motor coordination defect in the PCs specific Nova2 -cKO mouse. Rotarod test result at indicated age (weeks) (n>=5, **; p<0.01, ***; p<0.001). (D) PCs-degradation in the 12 months old PCs specific Nova2 -cKO mouse. tdTomato immunostaining images with anti-mRFP antibody in 4 weeks old or 12 months old Control tdTomato; Pcp2-Cre and Nova2-cKO tdTomato; Pcp2-Cre mouse. (E) The climbing fiber innervation defect and reduced PCs molecular layer thickness upon PCs specific Nova2 deficiency. vGlut2 ( green , climbing fiber terminal marker), calbindin D28 ( red , PCs marker), and NeuN ( blue , GCs marker) immunofluorescent staining images in the 16 weeks old Control Pcp2-Cre and Nova2 -cKO Pcp2-Cre mouse (upper left ). Quantification analysis of vGlut2, calbindin D28, and NeuN fluorescent intensity in the 16 weeks old Control Pcp2-Cre and Nova2-cKO Pcp2-Cre mouse (n=3, ***; p<0.001) ( upper right ). Quantification analysis of GC and PC-ML layer thickness in 4W or 16W Control Pcp2-Cre and Nova2-cKO Pcp2-Cre mouse ( lower panel ) (n=3, *; p<0.05, **; p<0.01). PC-ML: Purkinje cell and molecular layer. GC: granule cell layer. (F) Reduced PCs spine number upon the NOVA2 depletion. Golgi staining images of 16 weeks old Control Pcp2-Cre and Nova2-cKO Pcp2-Cre mouse ( left ). Quantification of spine numbers per 10 nm dendrite length (neurons number >=14 from three biological replicates) ( right ). “See also and Movie S1.”

Article Snippet: Primary antibodies used for CLIP, immunofluorescent staining (IF), and western blots (WB) were: mouse anti-GFP ( , clones 19F7 and 19C8, cTag-CLIP), rat anti-GFP (nacalai tesque, GF090R, IF: 1/1,000), mouse anti-GFP (Santa Cruz, sc-9996, WB: 1/1,1000), rabbit anti-mRFP/ tdTomato (ROCKLAND, 600–401–379, IF: 1/2,000), rabbit anti-NeuN/RBFOX3 (Millipore, ABN78, IF: 1/1,000), guinea pig anti-NeuN/RBFOX3 (Millipore, ABN90P, IF: 1/1,000), goat anti-NOVA2 (Santa Cruz, sc-10546, IF; 1/500, WB; 1/2,000, CLIP), rabbit anti-NOVA1 [EPR13847] (abcam, ab183024, IF: 1/1,1000), human anti-pan NOVA (anti-NOVA paraneoplastic human serum, WB: 1/5,000), rabbit anti-PTBP2 ( , IF:1/5,000, CLIP), guinea pig anti-vGlut1 (Synaptic System, 135 304, IF:1/2,000), rabbit anti-vGlut2 (Synaptic System, 135 403, IF:1/2,000), mouse anti-VGAT (Synaptic System, 131 011, IF: 1/1,000), rabbit anti-Calbindin D28 (Millipore, AB1778, IF: 1/1,000), goat anti-Calbindin D28 (Santa Cruz, sc-7691, IF: 1/500), rabbit anti-ITPR1/InsP3R, type1 (Millipore, ABS55, IF:1/2,000), mouse anti-GAPDH (Santa Cruz, sc-32233, WB: 1/1,000).

Techniques: Marker, Staining, Immunostaining