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. 2020 Aug;26(8):1038–1048. doi: 10.1261/rna.075275.120

FIGURE 4.

FIGURE 4.

Presence of m5C modification on EBER1 elicits a reduction in RNA levels in vivo. (A) Five BJAB-B1 clones generated by CRISPR knockout of NSUN2 (cl.1–cl.5) were probed by northern blot for EBER1. Please see Supplemental Figure 1 for further characterization of knockout cell lines. U6 was probed as a loading control. Quantification of a representative northern blot is shown in the graph below. In the absence of m5C modification, EBER1 levels are elevated approximately two- to threefold. (B) CRISPRi against ANG in wild-type or NSUN2 knockout BJAB-B1 cells. ANG mRNA and EBER1 levels were measured by qRT-PCR following ANG knockdown. A representative northern blot probing for EBER1 is also shown. ANG depletion in wild-type cells results in an increase in EBER1 levels, but not in NSUN2 knockout cells. Values are the average of three independent biological replicates; error bars indicate standard deviation. (C) Wild-type or NSUN2 knockout BJAB-B1 cells were treated with the ANG inhibitor N-65828 at 100 µM for the indicated time periods. A representative northern blot analysis probing for EBER1 and U6 as a loading control is shown. The graph underneath depicts the EBER1 levels measured by qRT-PCR normalized to GAPDH expression. Values are the average of three independent biological replicates; error bars indicate standard deviation. In wild-type cells, inhibition of ANG results in a statistically significant increase in EBER1 levels (P-values from Student's t-test are indicated), while in NSUN2 knockout cells EBER1 abundance does not change significantly. (D) Stress-induced ANG-mediated cleavage of EBER1 is enhanced by m5C modification. EBV-positive wild-type and NSUN2 knockout cells were treated with 0.5 mM arsenite for the indicated time periods followed by northern blot analysis to quantify EBER1 and tRNA levels. While tRNAGLY is less stable upon stress in NSUN2 KO cells compared to wild-type cells, EBER1 levels show the opposite trend by being largely unaffected when unmodified and prone to degradation in the presence of m5C. Quantification of EBER1 and tRNAGLY northern blots normalized to 5.8S rRNA levels, which remained unchanged upon arsenite treatment, from three independent biological replicates is shown; error bars indicate standard deviation.