3' RACE-seq of endogenous LINE-1 mRNA's 3' non-templated ends in PA-1 control kd, XRN1 kd, DCP2 kd, and DIS3L2 kd (kd by RNAi for 48h = set 1, or 126h = set 2), and 293T wild-type and XRN1 KO.
Human LINE-1 retrotransposons can mobilize in the human genome by a copy-and-paste machanism involving RNA intermediates. LINE-1 mRNA 3' ends play a major role in initiation of reverse transcription (a step retrotransposition). To assess the impact of RNA 3' end dynamics of LINE-1 mRNA on its retrotransposition potential in human cells we analyzed 3' non-templated ends on LINE-1 mRNA by 3' RACE-seq. RNA was retrieved from multiple clonal 293T and PA-1 cells wild-type or knock-out in either XRN1, DCP2, or DIS3L2. Publication: D Janecki, R Sen, N Szóstak, A Kajdasz, M Kordys, K Plawgo, D Pandakov, A Philips, Z Warkocki (2024): LINE-1 mRNA 3' end dynamics shape its biology and retrotransposition potential. Nucleic Acids Research. https://doi.org/10.1093/nar/gkad1251 Overall design: 3' RACE-seq on endogenous LINE-1 mRNA from a total of 7 clonal wild-type 293T cells, 7 clonal XRN1 KO cell lines in 293T, 1 clonal DCP2 KO cell line in 293T, 1 clonal DCP2 plus XRN1 KO cell line in 293T. Also sequencing of artificial spike-ins with a known number of 0, 8, 16, 32, 64, and 128 consecutive A residues. All RACE-seq libraries were performed in 3 technical replicates for each mRNA analyzed. Note a set of 4 cellular conditions: wild-type, XRN1 KO, DCP2 KO, and DCP2 plus XRN1 KO (based on cells obtained from dr Sarah Slavoff, Luo et al., 2020, 10.1021/acs.biochem.0c00069) was performed only for reporter LINE-1 and GAPDH, and not the other 2 mRNAs. Libraries generated as described in Warkocki et al., 2018 (https://doi.org/10.1016/j.cell.2018.07.022) but PCR was done using the TaKaRa GLX polymerase.