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. 2024 Sep 11;15:7935. doi: 10.1038/s41467-024-51457-9

Fig. 6. Overexpression of RNase H1 represses RNA-mediated DNA double-strand gap repair.

Fig. 6

a Result of Western blot for human RNase H1 overexpression (OX) in HEK-293T cells, N = 2. Control, overexpression of a non-functional mutant of RNase H1. b Pie charts showing frequencies of sequencing reads displaying intron retention or pop-out following a gap by the sgRNAs A and B in the Sense and BranchΔ constructs with Control (left) and with HI OX (right). Percentages represent an average of 4 repeats with standard deviation in parenthesis; N = 4. The percentages of sequences with and without intron are bolded. *p = 0.029 comparing frequencies of the BranchΔ with those of the Sense construct via the two-tailed Mann–Whitney U test. c Sense/BranchΔ frequency ratios for two types of repaired sequences, with intron (green) and without intron (blue), detected in the sequencing libraries following a double-strand gap by the sgRNAs A and B in Control and H1 OX. d Boxplot showing the ratios of MMEJ frequencies between the Sense and the BranchΔ constructs of Control and H1 OX from Exon1-Exon2 microhomologies located upstream and downstream of the DNA gap. Twelve Exon1-Exon2 microhomologies (black dots) are shown for MMEJ following the DNA gap in the sense constructs generated by the sgRNA A and B. The thick red arrow indicates a ratio from the EE2R_GAAG microhomology (Supplementary Fig. 11b). The thin yellow arrow indicates a ratio from the EE12R_GTC microhomology (Supplementary Fig. 11b). For details see legend of Fig. 3c. e MMEJ frequencies from the outlier-data points presented in (d) derived from the exon-exon EE2R_GAAG microhomologies (thick, red arrows) that are the most distant from each other, and the EE12R_GTC microhomologies (thin, yellow arrows) that are the closest to each other (see Supplementary Figs. 11b and 12a), following a double-strand gap by the sgRNAs A and B in the Sense (red) and BranchΔ (green) constructs of Control and H1 OX. For (c) and (e), plotted data are the mean ± s.d. of the 4 biological replicates with the individual values shown as dots; N = 4. *p = 0.029 (two-tailed Mann–Whitney U test). Source data are provided as a Source Data file.