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. 2021 Jul 15;118(29):e2026813118. doi: 10.1073/pnas.2026813118

Fig. 6.

Fig. 6.

RMRP prevents lysosomal proteolysis of SNRPA1 by perturbing CMA. (A and B) SNRPA1 protein is stabilized by the autophagy inhibitor CQ but not by the proteasome inhibitor MG132. HCT116 p53+/+ (A) and H460 (B) cells were treated with MG132 (20 μM, 6 h) and CQ (50 μM, 8 h) before harvested for IB. (C) The presence of a KFERQ-like motif, LKERQ, in SNRPA1 and mutation of this motif to LKEAA. (D) Exogenous SNRPA1 interacts with exogenous HSPA8 via the LKERQ motif. HCT116 p53−/− cells were transfected with combinations of plasmids encoding Myc-HSPA8, Flag-SNRPA1, and Flag-SNRPA1-Mut followed by co-IP–IB assays. (E) Exogenous SNRPA1 interacts with exogenous LAMP2A. HCT116 p53−/− cells were transfected with plasmids encoding Flag-SNRPA1 and Myc-LAMP2A followed by co-IP–IB assays. (F) Overexpression of LAMP2A reduces the protein level of SNRPA1 in HCT116 p53+/+ cells. (G) Knockdown of LAMP2A increases the expression of SNRPA1 in HCT116 p53+/+. (H) The cellular distribution of SNRPA1 protein in HCT116 p53+/+ cells. GAPDH and Lamin B indicate the cytosolic and nuclear fractions, respectively. (I) CRISPR-Cas9–mediated ablation of RMRP reduces the SNRPA1 level in the nucleus, while increases the cytosolic accumulation of SNRPA1, in HCT116 p53+/+ cells. (J) Knockout of RMRP enhances the endogenous interaction of SNRPA1 and HSPA8.