Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating Aldh1a3 pre-mRNA 3'-end processing
- University of Rome Tor Vergata, Italy
- Fondazione Santa Lucia, Italy
- Institute of Cell Biology and Neurobiology CNR, Italy
Figures
Figure 1
Sam68 is highly expressed in NPCs and decreases during differentiation.
(A) qPCR analysis of Khdrbs1 mRNA levels in the cortex of embryonic (E10.5-E15.5) and post-natal (P0-25dpp) mouse brain. Khdrbs1 relative expression was evaluated by △CT method using L34 expression …
Figure 2 with 1 supplement
Ablation of Khdrbs1 perturbs neurogenesis in the embryonic cortex.
(A) Schematic representation of the stratification of the VZ/SVZ, intermediate zone (IZ) and cortical plate (CP) in the E13.5 embryonic cortex. Molecular markers, expressed by the respective cell …
Figure 2—figure supplement 1
Ablation of Khdrbs1 perturbs neurogenesis in the embryonic cortex.
(A) Representative images of immunofluorescence analysis of BrdU (left panel) and SOX2(center panel) performed in E13.5 cortex of embryos treated with BrdU for 2 hr. Bar graphs represent (mean±SD) …
Figure 3 with 1 supplement
Differentiation of NPCs in post-mitotic neurons is accelerated in the Khdrbs1-/- mouse cortex.
(A) Diagram of the experimental design to investigate the fate on NPCs during neurogenesis. Pregnant females were treated with BrdU at E12.5, and embryos were collected after 2 days for …
Figure 3—figure supplement 1
Ablation of Khdrbs1 does not affect overall embryo development.
Weight analysis of Khdrbs1+/+, Khdrbs1+/- and Khdrbs1-/- mouse embryos at E13.5, 15.5 and E17.5. At least 5 embryos for each genotype were weighted. Statistical analysis was performed using the …
Figure 4 with 3 supplements
Sam68-/-NPCs lose stemness and are prone to differentiate in culture.
(A) Bright field images of Khdrbs1+/+ and Khdrbs1-/- NPCs cultured in proliferating condition for 3 days in culture. The insets show higher magnification of a selected neurosphere. Black arrowheads …
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Figure 4—source data 1
List of the genes upregulated and downregulated inSam68 knockout NPCs.
Yellow=upregulated genes; green=downregulated genes. NPCs, Neural progenitor cells.
- https://doi.org/10.7554/eLife.20750.009
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Figure 4—source data 2
List of exons regulated by SAM68 in mouse NPCs.
Green: genes downregulated at expression level; Yellow: genes up-regulated at expression level; White: genes unchanged at expression level. NPCs, Neural progenitor cells.
- https://doi.org/10.7554/eLife.20750.010
Figure 4—figure supplement 1
Khdrbs1-/- NPCs lose stemness and are prone to differentiate in culture.
(A) Bar graph reporting the analysis of the diameter of neurospheres formed by Khdrbs1+/+ and Khdrbs1-/- NPCs in 1–3 days of culture. N = 3; *p<0.05. (B) Cell death analysis of Khdrbs1+/+ and Khdrbs1…
Figure 4—figure supplement 2
Khdrbs1-/- NPCs-derived neurons display more complex morphology.
(A) Images representing a typical neuron obtained through the differentiation of Khdrbs1-/- NPCs (upper image) and the representation of the scheme used to quantify the complexity of its structure …
Figure 4—figure supplement 3
High-throughput analyses of the transcriptomic of Khdrbs1-/-NPCs.
(A) Analysis of the gene and exon expression in Khdrbs1-/- vs Khdrbs1+/+ NPCs reporting upregulated and downregulated events. (B) Overlap between the genes regulated at expression and splicing level …
Figure 5 with 1 supplement
Sam68 regulates the alternative 3'-end processing of ALDH1A3 pre-mRNA.
(A) Diagram of ALDH1A3 gene structure. Red and green squares represent exons, respectively, upregulated and downregulated in the microarray analysis. Proximal polyadenylation site (pPAS) and distal …
Figure 5—figure supplement 1
Sam68 regulates the alternative 3'-end processing of ALDH1A3 pre-mRNA.
(A) Diagramatic scheme of the Aldh1a3 gene reporting the up- (red) and down-regulated (green) exons identified by the microarray experiment. The bar graphs represent the results of qPCR analyses …
Figure 6 with 1 supplement
Sam68 promotes ALDH1A3 expression and activity.
(A) Western blot analysis of the expression of ALDH1A3 and Sam68 in lysates from Khdrbs1+/+ and khdrbs1-/- NPCs. Vinculin was used as loading control. The bar graph represent densitometric analyses …
Figure 6—figure supplement 1
Sam68 promotes ALDH1A3 expression and activity.
(A) Western blot analysis of ALDH1A3△ expression in Khdrbs1-/- NPCs treated with 10 µM proteasome inhibitor MG132 and 2 µM autophagy inhibitor Chloroquine. HEK293 cells transfected with pCI-ALDH1A3△ …
Figure 7 with 2 supplements
ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs.
(A) Representative images of Khdrbs1+/+ NPCs transfected with GFP vector and Khdrbs1-/- NPCs transfected with GFP and GFP-ALDH1A3 vectors, cultured for 3 days in proliferating condition. Scale bar …
Figure 7—figure supplement 1
ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs.
(A) Representative images of Khdrbs1-/- NPCs transfected with GFP vector and Khdrbs1+/+ NPCs transfected with GFP and GFP-ALDH1A3△ vectors, cultured for 3 days in proliferating condition. Scale bar …
Figure 7—figure supplement 2
ALDH1A3-dependent glycolytic metabolism rescues the self-renewal potential of Khdrbs1-/- NPCs.
(A) Evaluation of the relative lactate production of Khdrbs1+/+ and Khdrbs1-/- NPCs cultured under proliferating condition. (B) Relative lactate production in Khdrbs1+/+ or Khdrbs1-/- NPCs treated …
Figure 8
Sam68 modulates neurogenesis through regulating ALDH1A3 expression.
Schematic model of the function of Sam68 in the modulation of NPCs stemness. High Sam68 expression in NPCs of the neocortex insures high expression of ALDH1A3 protein and anaerobic glycolysis, thus …
Additional files
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Supplementary file 1
List of the oligonucleotides used as PCR primers and siRNAs in the study.
- https://doi.org/10.7554/eLife.20750.022
