Fig. 5: m⁶A modification is crucial for generating retrotransposition-competent L1 RNPs.

a Scheme of LEAP assay with description. Ultracentrifugation and purification cellular RNP (left part of scheme, cell lysate in red, and sucrose cushion in gray). LEAP reaction (right part of scheme, L1 ORF1p in yellow, L1 ORF2p in magenta, 3′ RACE adapter in green). b Quantification of mRNA levels in the RNP fraction of pL1-expressing HeLa cells. cDNA synthesis in the absence of reverse transcriptase (lane 2–4) and transfection of empty vector (lane 6) served as negative controls. The RT-PCR products of reporter L1 and GAPDH are of 158 and 106 bp, respectively (lane 6–8). Lane 1 and 5 show the DNA ladder. c Immunoblot assay of the RNP fraction from pL1-expressing HeLa cells. pan AGO served as a loading control. d LEAP assay using RNP fraction from pL1-expressing HeLa cells. The LEAP product is a diffuse band of 300–400 bp. The images of b–d are representative of three independent experiments. e A schematic of the L1-MS2 construct (pAD3TE1) carrying T7-tagged ORF1p (green) and MS2-stem-loops with Q670-labeled MS2 binding probes (red). f Immunofluorescence and RNA FISH images depicting HeLa cells transfected with pAD3TE1 L1Hs (top) or L1 m⁶A mut (bottom). Images for T7-tagged ORF1p (green), L1-MS2 RNA (red), and the merged images with DAPI (blue) are indicated. The images are representative of two independent experiments. g The number of L1 RNP foci in pAD3TE1-expressing HeLa cells. Colocalizing puncta within an intermolecular distance of 330 nm were counted as L1 RNP using z-stack analysis. h Intensity of L1 ORF1p in colocalizing puncta. Each point represents the intensity of L1 ORF1p per cell. g, h Boxplots indicate median (red middle line), 25th, 75th percentile (gray box) and 5th and 95th percentile (whiskers). 43 cells for L1Hs and 34 cells for L1 m⁶A mut, two-sided Kolmogorov–Smirnov test, ****p < 0.0001. Source data are provided as a Source data file.