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. 2023 Jan 31;51(3):1326–1352. doi: 10.1093/nar/gkad033

Figure 7.

Figure 7.

DDX3X affects the stability of 3′-tracer tRNAs in vitro and in vivo. (A) Western blotting for DDX3X, DDX5 and eIF2α (serine-51 phospho) before and 48 h after transfection with siRNA pools (non-targeting as controls versus targeting each RNA helicase alone or in combination). Antibodies against vinculin were used as a loading control. Lane marked with (*) points at background eIF2α-P signal, while lanes marked with (**) indicate siRNA-mediated induction of eIF2α-P signal in RNA helicase knockdowns. (B) 3′-tracer tRNAs (8 nM final) were incubated with cytoplasmic protein extracts (2 or 4 μg) obtained from HeLa cells transfected with siRNAs targeting DDX3X or DDX3X in combination with DDX5. Reactions were separated by nPAGE and 3′ tsRNA signals were collected as described above. Black arrowhead, 3′-tracer tRNAs; green arrowhead, 3′ tsRNAs. (C) Western blotting for DDX3X in total protein extract obtained from BMDF after 72 h of 4-hydroxytamoxifen (4-OHT)-mediated deletion of the DDX3X gene locus. β-actin was used as a loading control. (D) NB using sequential probing of the same membrane against the 5′ and 3′ portions of tRNA-GlyGCC/CCC on total RNA purified from BMDF after 72 h of 4-OHT-mediated deletion of the DDX3X gene locus before and after As[III] exposure (0.5 mM for 1 h). Black arrowheads, mature tRNAs; red arrowhead, 5′ tsRNAs; green arrowhead, 3′ tsRNAs. (E) 3′-tracer tRNAs (8 nM final) were incubated with cytoplasmic protein extracts (5 or 10 μg) obtained from BMDF before or after 4-OHT-mediated deletion of the DDX3X. Reactions were separated by nPAGE and 3′ tsRNA signals were collected as described above. Black arrowhead, 3′-tracer tRNAs; green arrowhead, 3′ tsRNAs; grey arrowhead, signals from partial 3′ tsRNA degradation. (F) NB of total RNA purified from SEC fractions originating from As[III]-exposed BMDF before (upper panels), after 4-hydroxytamoxifen (4-OHT) mediated deletion of DDX3X (middle panels), and in the presence of ectopic expression of wild type DDX3X (pCMV-DDX3X, lower panels) using sequential probing of the same membrane. Probes for the 5′ (left panels) and 3′ (right panels) moieties of tRNA-GlyGCC/CCC were used. Black arrowheads, mature tRNAs; red arrowhead, 5′ tsRNAs; green arrowhead, 3′ tsRNAs.