Figure 5.

CDK5RAP1 Balances i⁶A and ms²i⁶A Concentrations in GICs in Response to the Microenvironment

(A) Mass spectrometry analysis of [i⁶A] and [ms²i⁶A] from the lysates of shControl- and shCDK5RAP1-, RFP-, mCdk5rap1-transfected JKGIC2 cells. Note that CDK5RAP1 knockdown induces an increase in the relative [i⁶A] in GICs, whereas CDK5RAP1 overexpression reduces the relative [i⁶A]. Each bar represents the SD value from three independent replicates. *p < 0.05.

(B) Schematic representation of the molecular characteristics of CDK5RAP1. Cysteine residues in the UPF0004 and radical SAM domains are crucial for the stabilization of the [4Fe-4S] clusters. The mitochondria localization signal (MLS) allows CDK5RAP1 to localize to the mitochondria. The TRAM domain is predicted to interact with tRNA species.

(C) The M.I. of ms²i⁶A corresponding to each mt-tRNA and tRNA expression level in JKGIC2 cells cultured in the presence of 21% and 1% O₂. The M.I.s in all CDK5RAP1-targeted tRNA species are increased under hypoxic conditions, but tRNA expression is not. The data are presented as the M.I. relative to the cells cultured under normoxia. Each bar represents the SD value from two independent replicates. For the procedure regarding the measurement of M.I., also see Figure S5D.

(D) Hypoxic conditions decrease intracellular [i⁶A] and increase the secretion of ms²i⁶A by JKGIC2 cells. The data are presented as the percentage of the levels under normoxia. Each bar represents the SD value from three independent replicates. *p < 0.05.

(E and F) Hypoxic conditions significantly increased the expression levels of VEGF mRNA (E) but not CDK5RAP1 mRNA (F) in JKGIC2 cells. Each bar represents the SD value from four independent replicates. *p < 0.05.

Also see Figure S5.