Fig 1: treRNA1 enhances ALKBH5 demethylase activity: (A) Commercially procured active recombinant ALKBH5 in the presence of an increasing concentration of HPLC purified treRNA1 demethylated non-immunized splenic B-cell mRNA. Purified GST was an experimental control. The results were normalized with total RNA and expressed as mean±SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc analysis. bp < 0.01, dp < 0.001 versus GST incubated (no ALKBH5) RNA. Dp< 0.001 versus ALKBH5 0.5μM (no treRNA1). (B, C) Quantification of m6A RT-qPCR (G) and HuR IP RT-qPCR of demethylated non-immunized splenic B-cell mRNA. Mock antibody/ GST protein was used as the experimental control. The results were first normalized with their respective loading controls (Luciferase mRNA), neutralized with the average of respective total RNA, and expressed as mean ± SD, set at 1. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. ap < 0.05 vs m6A enriched GST treated RNA (B) or HuR precipitated GST treated RNA (C). (D, G, N) Total RNA isolated from shTRERNA1 stably infected DLBCL cells were subjected to quantification of indicated genes with RT-qPCR. The results were normalized with GAPDH (reference gene), set to 1 for shSCR-infected cells, and expressed as mean ± SD. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. ap < 0.05, bp < 0.01, dp < 0.001 versus corresponding shSCR infected cells. (E, L) Protein lysates were harvested from indicated shTRERNA1 stably infected DLBCL cells and were analyzed using immunoblotting with the specified antibodies. shSCR infected cells were used as internal control; S: shSCR, T: shTRERNA1 (F, M) Densitometric quantifications of immunoblots of specified antibodies from Fig. E and L, respsectively. Values were first normalized with their respective loading controls and neutralized with the average of shSCR infected cells and expressed as mean ± SD, which was set at 1. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. ap< 0.05, bp< 0.01, cp< 0.005 versus corresponding shSCR infected cells. (H) Total RNA was collected from shTRERNA1 stably infected Farage cells along with shSCR infected cells and subjected to m6A quantification. The results were normalized with total RNA and expressed as mean±SD. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. dp < 0.001 versus corresponding shSCR-infected cells. (I, K) m6A IP and HuR IP enriched RNA were subjected to RT-qPCR. Normal mouse IgG (mock) (I) and purified GST protein (K) were used as experimental controls. The results were first normalized with their respective loading controls (Luciferase mRNA) and neutralized with the average of shSCR infected cells (enriched with m6A and HuR, respectively) and expressed as mean ± SD, which was set at 1. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. ap < 0.05, bp < 0.01, dp < 0.001 versus corresponding shSCR infected cells. (J) RT-qPCR of nuclear- and cytoplasmic-enriched fraction isolated from shDDX46 stably infected DLBCL and shSCR infected cells. The results were first normalized with their respective loading controls (Luciferase mRNA), neutralized with the average of shSCR infected nuclear fraction, and expressed as mean ± SD, set at 1. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc analysis. ap < 0.05, bp < 0.01, dp < 0.001 versus corresponding SCR nuclear fraction, Bp < 0.01, Cp < 0.005, Dp < 0.001 versus corresponding SCR cytoplasmic fraction, αp < 0.05, γp < 0.005, δp < 0.001 versus corresponding shDDX46 nuclear fraction. (O) Protein lysates were harvested from shTRERNA1 stably infected Toledo cells treated with either isotype or BCR mimetics and were analyzed using immunoblotting with the specified antibodies. shSCR infected cells were used as internal control.