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. Author manuscript; available in PMC: 2022 Mar 8.
Published in final edited form as: Cell Rep. 2022 Feb 8;38(6):110349. doi: 10.1016/j.celrep.2022.110349

Figure 5. A RLRKGR motif on PD-L1 protects TβRI mRNA from degradation by competing with the RNA exosome complex.

Figure 5.

(A) EXOSC10 was knocked down by two different shRNAs and the cells were collected for qRT-PCR (upper) and WB (lower) for TβRI. Knockdown of EXOSC10 led to increases of TβRI mRNA and protein level of HSCs. *p < 0.05, **p < 0.01 by ANOVA, n = 3.

(B) Left: control HSCs, HSCs with PD-L1 knockdown, EXOSC10 knockdown, or knockdown of both were collected for qRT-PCR and WB for TβRI. EXOSC10 knockdown rescued TβRI mRNA and TβRI protein of PD-L1 knockdown HSCs. ***p < 0.001 by ANOVA, n = 3. Right: the stability of TβRI mRNA was assessed in the presence of Actinomycin D. PD-L1 knockdown accelerated the degradation of TβRI mRNA in HSCs and this effect was abrogated by knockdown of EXOSC10. *p < 0.05 by ANOVA, n = 3.

(C) RIP assay revealed that knockdown of PD-L1 led to increased binding of TβRI mRNA to EXOSC10 in HSCs. ***p < 0.001 by ANOVA, n = 3.

(D) RIP assay revealed that PD-L1 FL and PD-L1 T + C competed off EXOSC10/TβRI mRNA binding promoted by PD-L1 knockdown in HSCs. ***p < 0.001 by ANOVA, n = 3.

(E) Left: the RLRKGR motif on PD-L1 T + C was changed to ALAAGA (PD-L1 T + C [4A]) or ALAAGR (PD-L1 T + C [3A]). RIP assay revealed that both mutants abrogated PD-L1 T + C/TβRI mRNA binding in HSCs. ***p < 0.001 by ANOVA, n = 5. Right: qRT-PCR and WB revealed that both mutants failed to rescue TβRI mRNA and TβRI protein of PD-L1 knockdown HSCs compared with wild-type PD-L1 T + C. ***p < 0.001 by ANOVA, n = 4.

(F) RIP assay showed that PD-L1 T + C competed off EXOSC10/TβRI mRNA binding in PD-L1 knockdown HSCs, and this effect was abrogated by either mutant. **p < 0.01, ***p < 0.001 by ANOVA, n = 3. All data are represented as mean ± SEM.