Extended Data Fig. 3: L1 restriction by DBR1 and DIS3.
From: LINE-1 transcription activates long-range gene expression
a, Western blotting showing the level of DBR1 and endogenous L1 ORF1p in the K562 cells infected with two independent Cas9/sgRNAs targeting DBR1 and two independent non-target control Cas9/sgRNAs, independently repeated once with similar results. HSP90 is loaded as control. b, Donut chart showing that DBR1-regulated L1s (P < 0.05, DESeq2 analysis) are predominantly localized in introns in K562. c, Size distribution of DBR1-regulated L1s (P < 0.05, DESeq2 analysis) and all genomic L1s (based on hg38 RepeatMasker annotation) in K562. P value, two-tailed Kolmogorov–Smirnov test. d, The read counts of total lariats and L1-containing lariats, obtained from the ribosome-depleted RNA-seq data by a lariat read-count analysis strategy81 and normalized against unmapped singlets in control and DBR1 knockout K562 cells. n = 2 biological replicates. The center value is the mean. e, A working model that DBR1 restricts intronic L1 expression by debranching lariats into linear RNAs that are subsequently degraded. In DBR1 mutant cells, the level of L1-containing lariats is elevated, and the L1 within the lariat is proposed to be translated into L1 proteins. f, Donut chart showing genomic distribution of the DIS3-restricted L1s (P < 0.05, DESeq2 analysis) in K562 cells. g, Density plot showing size distribution of the DIS3-regulated L1s (P value < 0.05, DESeq2 analysis) and all genomic L1s (based on hg38 repeatmasker annotation) in K562 cells. P value, two-tailed Kolmogorov–Smirnov test. h, Schematic of DIS3 domains and the two point mutations to inactivate the PIN and RNB domains, respectively21. i, RNA-seq showing the relative expression level of full-length L1s in DIS3 knockdown HEK293 cells, rescued with the intact DIS3 or DIS3 mutants with inactive PIN or RNB domains, as indicated. RNA-seq reads were unique-mapped to individual L1 locus. As indicated, the PIN domain with endonucleolytic activity accounts for the L1 restriction. j, Aggregated line plot showing average enrichment of the DIS3 PAR-CLIP signal over full-length L1s. Genome-wide mappability displays genome-wide 100-bp-short-read alignability (data from UCSC). The results indicate that DIS3 directly binds L1 RNAs.
