Skip to main content
. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Cell Stem Cell. 2022 Nov 15;29(12):1653–1668.e8. doi: 10.1016/j.stem.2022.10.009

Figure 3. U1 snRNA drives alternative polyadenylation and skews the balance toward long CD47 mRNA isoforms in aged muscle stem cells.

Figure 3.

(A) CD47 mRNA sequences upstream of polyadenylation site 1 (PAS1) from indicated species were aligned. A highly conserved U1 snRNA binding site (blue box) was identified upstream of PAS1 (red box). (B) Scatter plot shows the expression levels of U1 snRNA in sorted aged MuSCs relative to young MuSCs, measured by q-RT-PCR (mean ± SEM, n= 4 young and 4 aged mice, 2 independent experiments). Two-tailed paired t test. (C) Bar graph shows the relative abundance of CD47 mRNA with long 3’ UTR, as a fraction of the total CD47 mRNA, in young and aged MuSCs treated in vitro with AMOs to PAS1, U1 snRNA binding site on CD47 transcript (CD47 U1), and control (ctr) (mean ± SEM, 4 independent experiments). Two-way ANOVA analysis. (D, E) Surface CD47 protein expression was measured by flow cytometry in young and aged MuSCs treated in culture with AMOs to PAS1, CD47 U1 and control AMO. (D) Bar graph shows the proportion of young and aged MuSCs expressing surface CD47 upon treatment with PAS1 (red) or CD47 U1 (blue) AMO, relative to ctr AMO treated MuSCs (mean ± SEM, 3 independent experiments). Two-way ANOVA. (E) Bar graph shows the expression level of surface CD47 protein on young (left) and aged (right) MuSCs treated as described above, measured as MFI and quantified relative to control AMO treated MuSCs (mean ± SEM, 3 independent experiments). Two-way ANOVA. (F) MuSCs sorted from young and aged GFP/Luciferase mice were treated overnight with AMOs to CD47 U1 or control (ctr) AMO and transplanted (100 cells/injection) into the TA muscle of hindlimb-irradiated NOD/SCID mice. To monitor MuSC engraftment, mice were imaged weekly by BLI. Scatter plot shows the BLI signal intensity of the engrafted transplants, 3 weeks after transplantation. Line represents the median BLI signal with interquartile range (n= 14 mice, 2 independent experiments). Wilcoxon test. (G) Model for post-transcriptional regulation of CD47 in aged MuSCs. U1 snRNA, which is upregulated in aged MuSCs, binds to its site on CD47 3’UTR upstream of PAS1, blocks usage of CD47 PAS1 site, and shifts the balance toward the long isoform, leading to increased surface CD47 expression. Treatment of aged MuSCs with the CD47U1 AMO (intervention), which competes with U1 snRNA for its binding site, prevents alternative polyadenylation, leads to increased production of the short CD47 mRNA isoform and decreased surface CD47 expression, and improves MuSC engraftment to levels similar to those of young MuSCs. The speed dial shows that increasing levels of CD47U1 AMO shift the balance towards the short CD47 isoform and a more youthful molecular phenotype.