Journal: bioRxiv

Article Title: The AUUUC repeat RNA aggregates sequester RNA-binding proteins like NOVA2 and lead to iron dyshomeostasis in spinocerebellar ataxia type 37

doi: 10.1101/2025.09.03.673996

Figure Lengend Snippet: (A) Schematic representation of the NOVA2 Human Untagged Clone (#SC303210, OriGene) containing the full-length NOVA2 cDNA (NM_002516) that we modified by inserting a 14×His-tag in-frame at the C-terminus of the NOVA2 open reading frame. The scheme illustrates the NOVA2 coding sequence (yellow), the inserted 14×His-tag (orange), followed by the stop codon (red). (B) hNSC co-overexpressing the non-pathogenic N(ATTTT) 7 , N(ATTTT) 139 or the pathogenic Ins(ATTTC) 58 sequences and NOVA2-His-tag, show aberrant nuclear AUUUC repeat aggregates that colocalize with NOVA2 (white arrows), but not AUUUU repeat accumulation, after staining with a specific FISH probe followed by immunofluorescence with the Anti-his-tag antibody, visible at 48 hours post-transfection. GFP expression was used as a marker for transfection. Images acquired in Leica Microsystems TCS SP5 II confocal microscope, using a HC PL APO Lbl. Blue 63× /1.40 Oil objective with 8× zoom. Scale bar = 2 µm.

Article Snippet: To create the NOVA2-His-tag expression vector, the NOVA2 Human Untagged Clone (#SC303210, Origene) vector was modified by a His-tag sequence of 14 histidines inserted, in frame, at the C-terminal of the NOVA2 by the in vivo assembly (IVA) cloning method ( ).

Techniques: Modification, Sequencing, Staining, Immunofluorescence, Transfection, Expressing, Marker, Microscopy

Journal: bioRxiv

Article Title: The AUUUC repeat RNA aggregates sequester RNA-binding proteins like NOVA2 and lead to iron dyshomeostasis in spinocerebellar ataxia type 37

doi: 10.1101/2025.09.03.673996

Figure Lengend Snippet: (A) Representative high-quality scans of HEK293T cells were analyzed under each experimental condition: cells co-expressing either the SCA37 pathogenic repeat insertion, Ins(ATTTC) 58 , or the non-pathogenic N(ATTTT) 7 and NOVA2-His-tag; cells expressing the non-pathogenic N(ATTTT) 7 and NOVA2-His-tag sequence, but with no NiCl supplementation; and mock-transfected cells (expressing no plasmids). The fluorescence images highlight the localization and expression patterns of iron (Fe) and nickel (Ni) under each condition. Zinc (Zn) delineates the nucleus and potassium (K) the cytoplasm. The X-Ray beam size was approximately 35 nm (horizontal) × 38 nm (vertical) at an excitation energy of 17.1 keV, with an X-Ray flux of 2×10 11 photons.s -1 . A dwell time of 50 ms.pixel −1 and a stepsize of 50 nm were used. (B) Pearson’s Correlation Coefficient for iron and nickel distribution was calculated for individual cells under each experimental condition. Statistical significance between the conditions was determined using a Mann-Whitney test, with a threshold of p<0.05 for significance. (C-F) Graphical representation of the concentration of (C) iron and (D) nickel, in the whole cell; and concentration of (E) iron and (F) nickel, in the nucleus. For (C), (D), (E) and (F), statistical significance was determined using One-Way ANOVA, with p<0.05 considered significant. Scale bar = 50 pixels.

Article Snippet: To create the NOVA2-His-tag expression vector, the NOVA2 Human Untagged Clone (#SC303210, Origene) vector was modified by a His-tag sequence of 14 histidines inserted, in frame, at the C-terminal of the NOVA2 by the in vivo assembly (IVA) cloning method ( ).

Techniques: Expressing, Sequencing, Transfection, Fluorescence, MANN-WHITNEY, Concentration Assay