Endogenous ERV expression in murine P19 cells upon transfection with tRF3b oligonucleotides
Description
To confirm a direct effect of 3’-tRFs on protein levels of endogenous ERVs, we transfected P19 mouse embryonic teratocarcinoma cells with synthetic 3’-tRF oligonucleotides and observed reduced levels of MusD- and IAP-Gag-related proteins. For MusD, previous studies from the Heidmann lab using a plasmid-expressed MusD element detected several MusD Gag-reactive products corresponding to the MusD-encoded protease-mediated cleavage of the Gag polyprotein, with apparent molecular weights of approximately 26, 36, 43, and 64 kDa (Ribet et al., 2004). In the endogenous P19 cell lysates analyzed here, we detect bands corresponding in apparent molecular weight to these previously described MusD Gag products, including the ~26 kDa MusD Gag-related product (highlighted in red) shown in the main manuscript. In addition, the endogenous P19 blot contains further MusD-reactive bands. These additional bands most likely represent the expression of multiple endogenous MusD or MusD-like ERV elements in P19 cells, which may encode Gag proteins with slightly different sizes and/or processing patterns. The absence of these bands from the human HeLa negative-control lane supports that they are specific to endogenous murine ERVs. For the MusD blot, samples were loaded in duplicate from left to right using the following 3’-tRF oligonucleotides: lane 1-2) Lys-TTT-m1 tRF3b (rUrCrA rArGrU rCrCrC rUrGrU rUrCrG rGrGrC rGrCrC mA); lane 3-4) NT tRF3b control (rUrUrG rArUrG rUrGrU rUrUrA rGrUrC rGrCrU rArCrC mA); lane 5) HeLa negative control; lane 6-9) unrelated MusD samples, not used. For IAP, the major Gag protein p73 has been described previously (Poznanski and Calarco, 1991), and later studies further showed that IAP Gag antibodies detect additional Gag-related products, including the p41 partially processed Gag species (Ribet et al., 2008; Robbez-Masson et al., 2018). In the endogenous P19 cell lysates analyzed here, we detect IAP-reactive bands corresponding in apparent molecular weight to the previously described p73 and p41 IAP Gag-related products. Additional IAP-reactive bands are also observed, including the ~26-30 kDa IAP Gag-related products (highlighted in red) shown in the Extended Data section of the study. These additional bands likely reflect expression of multiple endogenous IAP or IAP-like ERV elements in P19 cells, which could encode Gag proteins with slightly different sizes and/or processing patterns. The absence of these bands from the human HeLa negative-control lane supports that they are specific to endogenous murine ERVs. For the IAP blot, samples were loaded in duplicate from left to right using the following 3’-tRF oligonucleotides: lane 1-2) NT tRF3b control (rUrUrG rArUrG rUrGrU rUrUrA rGrUrC rGrCrU rArCrC mA); lane 3-4) Phe-GAA-m1 tRF3b (rUrCrG rArUrC rCrCrG rGrGrU rUrUrC rGrGrC rArCrC mA); lane 5) HeLa negative control; lane 6-9) unrelated IAP samples, not used.
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Steps to reproduce
P19 mouse embryonic teratocarcinoma cells (ATCC, CRL-1825; 150,000 cells per well) were seeded in 6-well plates one day prior to transfection with 200 pmol synthetic 3’-tRF oligonucleotides (Integrated DNA technologies) using Lipofectamine RNAiMAX (Invitrogen; 13778075). Protein was harvested 24 hours post-transfection. Cells were washed with ice-cold PBS pH 7.2 (Gibco; 20012027) and lysed in ice-cold 1x Laemmli buffer (50 mM Tris-Cl pH 6.8, 10% glycerol, 1% SDS, and 4 M urea). DTT (50 mM) and bromophenol blue (0.05% w/v) were added to the lysates before they were heated for 10 minutes at 95°C and centrifuged at 14,000 rcf for 20 minutes at 4°C. Proteins were separated by 12% SDS-PAGE alongside protein size standards (Bio-Rad; 1610374) and transferred onto a 0.45 μM nitrocellulose membrane (Bio-Rad; 1620115) using the Mini Trans-Blot system (Bio-Rad; 1703930). Transfer efficiency was checked by staining with Ponceau S (0.1% w/v) in acetic acid (5% v/v). Membranes were blocked in 5% Blotto non-fat dry milk (Santa Cruz Biotechnology; sc-2324) in 1xTBS with 0.1% Tween-20 (Thermo Scientific; J20605-AP) for 1 hour. All antibody incubations were performed in 0.5 % Blotto non-fat dry milk in 1xTTBS. Rabbit antiserum raised against MusD Gag (GenScript Custom Order of antiserum against the MusD1 polypeptide specified in Ribet et al., 2008) diluted 1:1,000 and IAP 'retrovirus-related Gag antibody' (Thermo Fisher, PA5-120018, polyclonal rabbit) diluted 1:1,500 in 1x TTBS with milk were used to detect MusD and IAP Gag proteins respectively overnight at 4C. Anti-rabbit HRP-coupled IgG was used as secondary antibody (Jackson Immuno Research, 111-035-144). Images were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106) and a Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad, 33925). To control for equal loading, membranes were stripped with Restore Stripping Buffer (Thermo Fisher Scientific, 21059) and reprobed for ACTB (Actin beta, Rabbit, Abcam, ab8227).
Institutions
- Cold Spring Harbor LaboratoryNew York, Cold Spring Harbor