FIG 4.

Northern blotting assays H₂O₂ treatment-caused tRNAs abundance changes in S. oligofermentans and overexpression of So-RNaseZ on tRNAs processing. (A) The anaerobically grown mid-exponential-phase wild-type (WT, upper panel) and So-RNaseZ-overexpressing (RNZ; lower panel) cells were treated with or without 500 μM H₂O₂ for 30 min and then collected for total RNA extraction. Northern blotting was used to examine the mature and precursor levels of three representative tRNAs using probes targeting the corresponding mature and 3′ trailer tRNA sequences as indicated at the gel tops, respectively. Total RNA of 0.5 μg and 10 μg was loaded for detecting mature tRNAs and precursors, respectively. 5S rRNA hybridization was used as a loading control. P, 40 ng of in vitro transcribed respective tRNA precursor fragment, composed of mature and 3′ trailer tRNA sequence, was loaded as a positive control. Black arrow indicates the position of the tRNA^Ala precursor, and RNA molecular size markers are shown on the side. Triplicate experiments were performed and representative images are shown. (B) Northern blotting signal intensities of RNA bands in H₂O₂-treated and -untreated samples in panel A were measured using Image J. The bar diagrams show the levels of mature tRNA (left) and tRNA precursors (right) in H₂O₂-treated and -untreated samples. No tRNA precursors were detected in So-RNaseZ-overexpressing cells treated with or without H₂O₂. The averages ± SD from triplicate experiments are shown. *, significantly different from the level of the corresponding tRNA species in anaerobically grown cells (Student’s t test; P < 0.05).