Fig 2: The CAUC motifs within the polypyrimidine tract upstream of CD44 v5 are also required for efficient splicing of exon v5. (A) Upper panel: schematic representation of CD44 exon v5 mut3 and mut4 constructs. The labels are the same as in Figure 4B. Lower panel: RT-PCR analysis of in vivo splicing pattern of wild-type, mut3 and mut4 constructs transfected into HEK 293 cells. (B) 32P-labeled RNA transcripts containing 70 nt intron sequence upstream of exon v5 and the first 16 nt of exon v5 were incubated with HeLa nuclear extract and subjected to UV irradiation and RNase digestion. The crosslinked proteins (lanes 1–4) were immunoprecipitated with anti-U2AF65 (lanes 5–8), anti-YB-1 (lanes 9–12) or a control antibody (lanes 13–16). The immunoprecipitates were analysed by SDS-PAGE and visualized by autoradiography. 10% equivalents of the inputs were loaded directly (lanes 1–4). (C) RT-PCR analysis of splicing activity of the wild-type, mut2, mut3, mut4 and mut2 + 3 minigene constructs in MDA-MB-231 cells stably transfected with control (luciferase) shRNA (lanes shLuc), YB-1 shRNA (lanes shYB-1) or co-transfected YB-1 shRNA together with a YB-1-resistant construct (4 µg, lanes shYB-1 + R). The averages of exon inclusion percentage with standard deviations are shown below (n = 3). The expression level of YB-1 was detected by western blotting, and GAPDH served as a loading control.

Fig 3: The effect of intranasally injected YB-1 on learning and spatial memory of 6-months-old 5XFAD mice as shown by tests in the Morris water maze. A, A positive effect of intranasally injected YB-1 on learning abilities of transgenic 5XFAD mice (Tg+YB-1) as shown by training trials in the Morris water maze. Abscissa, the training period (days); ordinate, the latent period of finding the invisible save platform (s). B, Duration of staying in the maze sectors for the 1 min probe trial period. C, Frequency of visiting the maze sectors for the 1 min probe trial period. Abscissa, categorized groups of experimental animals. Hatched columns represent the target sector. Open columns represent indifferent sectors of the water maze. According to the Post hoc analysis with LSD test, significant differences *p <0.05; **p <0.01; ***p <0.001.

Fig 4: The effect of intranasally-injected YB-1 on temporal dynamics of plaque density in the brain of transgenic 5XFAD mice. Abscissa, age of mice (month); ordinate, plaque density (n/mm2) in view field (x10). Filled columns represent data for 5XFAD mice intranasally administered with YB-1; open columns represent data for control Tg mice injected with saline; hatched columns represent data for Tg+BSA mice. A,B, and C, Plaque densities in the cortex and hippocampal areas CA1 and CA3, respectively. D,E, and F, representative microphotographs of the cortex, CA1, and CA3 respectively, for Tg+saline (left column) and Tg+YB-1 (right column) mice. The data are given as m±SEM. Significant differences for Tg+saline mice in each age group are denoted by asterisks (*): ***p<0.001; **p<0.01; *p<0.05.

Fig 5: Penetration of Cy3-labeled YB-1 into primary neuronal culture of rat hippocampus.The culture cells were incubated with Cy3-labeled YB -1 or Cy3-labeled BSA for 2 h. Then cell lysate proteins were separated by SDS-PAGE and detected for fluorescence (A) and Coomassie brilliant blue staining (B). Lanes: 1, molecular mass markers with fluorophore-labeled 72 kDa marker (Thermo Scientific); 2, Cy3-labeled BSA; 3, Cy3-labeled YB-1; 4, Cell lysates of the neuronal culture after 2 h incubation with Cy3-labeled BSA; 5, Cell lysates of the neuronal culture after 2 h incubation with Cy3-labeled YB-1.