3' RACE-seq of reporter LINE-1 and control mRNAs' 3' non-templated ends in 293T 'wild-type', XRN1 KO, DCP2 KO, DCP2 plus XRN1 KO cells
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, and control GAPDH and PABPC4 mRNAs by 3' RACE-seq. RNA was retrieved from multiple clonal 293T cells wild-type or knock-out in eitehr XRN1, DCP2, or both (DCP2 plus XRN1). 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 reporter LINE-1, GAPDH, PABPC4, and ACTB mRNAs 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. 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 in 3 technical replicates. 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.