Fig. 3: L1 biochemical activities, priming and cytoplasmic reverse transcription of L1.
From: Structures, functions and adaptations of the human LINE-1 ORF2 protein
a, Denaturing gel ORF2p RT assay. ORF2p core was an efficient DNA polymerase on all template–primer combinations; RNA priming on an RNA template was reduced but remained significant, with time-dependent full template-length (FTL) reaction products. NTA (+) and template jumping/switching (##) larger products were clearer on longer exposure (Extended Data Figs. 3–5 and Supplementary Figs. 3 and 4). b, ORF2p core (33 nM) single dATP incorporation kinetics with RNA or DNA template and 20 nt DNA primer. c, Extension of very short (5–10 nt) primers, pre-annealed to DNA or RNA templates, by ORF2p and HIV-1 RT; n = 4 (DNA), n = 3 (RNA) independent samples over two experiments. d, ORF2p RT assay showing efficient elongation of an RNA hairpin to FTL; HIV-1 RT showed minimal elongation. e, ORF2p efficiently extended a uridylated Alu-derived RNA hairpin. Ribonucleoside triphosphate incorporation was strongly selected against. f, Immunofluorescence of HeLa cells transfected for 24 h with WT or mutant L1 constructs (ORFeus-Hs) stained for RNA:DNA hybrids with catalytically inactive RNase H1 (dRNH1) and ORF2p (Flag). Cytosolic RNA:DNA hybrids colocalized with ORF2p, depended on RT activity, were ablated by 50 µM d4T and did not depend on EN activity, ruling out a nuclear origin. Hybrids were most prominent in L1 granules but were still present when ORF1p was removed (ORF2 only, monocistronic). g, Top left, ORF1p induction by 1 µM decitabine in THP1 monocytes. Concomitantly, interferon (IFN) production increased (secreted luciferase reporter, top right; lum., luminescence), further augmented by knockout of TREX1, a nuclease that degrades L1 cDNA. Bottom: treatment of these cells with 10 µM cGAS inhibitor G140 or 50 µM d4T RTI reduced baseline and decitabine-induced IFN production; 10 µM POC d4T, a more efficiently triphosphorylated d4T prodrug, reduced IFN further. For IFN, n = 4 biologically independent samples over two experiments. Scale bars, 10 μm. All error bars indicate s.d.
