Fig. 1: Identification of an ORF1p RNA binding mutant critical for L1 retrotransposition and ORF1p cytoplasmic foci formation.

a Schematic of a full-length RC-L1 (L1.3: Genbank Accession #L19088). ORF1p functional domains are noted below the schematic and include the coiled-coil domain, the RNA recognition motif (RRM), and carboxyl-terminal domain (CTD). Green arrowhead, position of the in-frame FLAG epitope tag. Open triangle, relative position of a triple mutant (R206A/R210A/R211A) in the RRM domain. b WT ORF1p and the ORF1p-FLAG R206A/R210A/R211A mutant are stably expressed in HeLa-JVM cells. Western blot with an anti-FLAG antibody. A construct lacking the FLAG epitope tag (pJM101/L1.3 [no FLAG]) served as a negative control. GAPDH served as a sample processing control. c Schematics of the retrotransposition indicator cassettes used in this study. A retrotransposition indicator cassette (REP) was inserted into the 3′UTR of an L1 in the opposite orientation relative to sense strand L1 transcription. The REP gene contains its own promoter (upside down arrow) and polyadenylation signal (open lollipop). The REP gene is interrupted by intron in the same orientation relative to sense strand L1 transcription. This arrangement ensures that REP expression only will occur if the sense strand L1 transcript is spliced and successfully integrated into genomic DNA by retrotransposition (bottom schematic, open triangles, target site duplications that typically are generated upon L1 retrotransposition). Three retrotransposition indicator cassettes are shown at the right of the figure: mneoI, which confers resistance to G418; mblastI, which confers resistance to blasticidin; and mEGFPI, which leads to enhanced green fluorescent protein (EGFP) expression. d Results of a representative mneoI-based retrotransposition assay. HeLa-JVM cells were co-transfected with phrGFP-C (transfection control) and either pJM101/L1.3FLAG (WT) or pALAF008 (M8 [RBM]). X-axis, L1 construct names, and representative retrotransposition assay results. Y-axis, relative retrotransposition efficiency; the number of G418 resistant (retrotransposition-positive) foci was normalized to the transfection efficiency (i.e., the percentage of hrGFP-positive cells). Pairwise comparison relative to the WT control: p = 2.1 × 10⁻¹²***. e The ORF1p-FLAG R206A/R210A/R211A mutant (M8 [RBM]) reduces the number of ORF1p cytoplasmic foci. Representative immunofluorescence microscopy images of U-2 OS cells expressing either WT ORF1p-FLAG (pJM101/L1.3FLAG) or ORF1p-FLAG R206A/R210A/R211A mutant (pALAF008 [M8 (RBM)]). The U-2 OS cells also expressed a doxycycline-inducible (Tet-On) mCherry-G3BP1 protein. White scale bars, 5 µm. f Quantification of immunofluorescence assays in U-2 OS cells. X-axis, L1 construct names. Y-axis, percentage of transfected cells containing ORF1p cytoplasmic foci. The number (n) inside the green bars indicates the number of individual cells counted in the assay. Pairwise comparisons relative to the WT control: p = 7.5 × 10⁻¹¹***. g RNA-immunoprecipitation (RNA-IP) reveals an L1 RNA binding defect in the ORF1p-FLAG R206A/R210A/R211A mutant (M8 [RBM]). HeLa-JVM cells were transfected with either pJM101/L1.3 (no FLAG), WT ORF1p-FLAG (pJM101/L1.3FLAG), or the ORF1p-FLAG R206A/R210A/R211A mutant (pALAF008 [M8 (RBM)]). An anti-FLAG antibody was used to immunoprecipitate ORF1p-FLAG; reverse transcription-quantitative PCR (RT-qPCR) using a primer set (L1 [SV40]) that amplifies RNAs derived from the transfected L1 plasmid was used to quantify L1 RNA. X-axis, constructs name. Y-axis, the enrichment of L1 RNA levels between the IP and input fractions. Blue rectangles, relative levels of control GAPDH RNA (primer set: GAPDH). Gray rectangles, relative levels of L1 RNA. In panels (d), (f), and (g), values represent the mean ± the standard error of the mean (SEM) of three independent biological replicates. The p-values were calculated using a one-way ANOVA followed by Bonferroni–Holm post-hoc tests; *** p < 0.001.