Fig 2: RNA-Dependent Association of TUT4 and TUT7 with MOV10(A) Mass spectrometry of coIPs with EGFP-TUT4 (6) and controls (6). Normalized mean intensity (semiquantitative measure of protein abundance) and specificity (quotient of mean intensity in EGFP-TUT4 coIP and in control coIP) are plotted. Only hits identified in at least 3 of 6 EGFP-TUT4 coIPs are shown.(B) Mass spectrometry of coIPs with EGFP-TUT7 (7) depicted like in (A). Only hits identified in at least 4 of 7 EGFP-TUT7 coIPs are shown.(C) Mass spectrometry of coIPs with EGFP-MOV10 (7) depicted like in (A). Only hits identified in at least 3 of 7 EGFP-MOV10 coIPs are shown.(D) Flowchart of experiments used to study RNA-dependence and stability of the TUT4 and TUT7 interactions with MOV10 (left panel) and results of the respective experiment (right panel). CoIP was done with EGFP-MOV10 as bait. Lanes 1–4: input and coIP with lysates from control EGFP-expressing cells; lanes 5–13: coIP with lysates from EGFP-MOV10-expressing cells; lanes 5–8: input proteins; lanes 9–13: enriched proteins without (lanes 9 and 11–13) or with RNase A (lane 10), washed with increasing salt concentrations as indicated (lanes 11–13). Supernatants after incubation with (lane 14) or without (lane 15) RNase A. Blots were probed for MOV10, TUT4, TUT7, PABPC1, and GAPDH as indicated. Probing for GAPDH and control coIP was done to show the absence of non-specific interactions.(E) TUT7 coIP with RNA after in vivo UV-crosslinking using monoclonal anti-FLAG antibodies and lysates from cells expressing reporter L1 RNAs and either FLAG-TUT7 or control MBP-TUT7. Enrichment fold was calculated by 2ˆΔΔCt method, dividing enriched L1 or GAPDH mRNAs in FLAG-TUT7 coIP by their amounts non-specifically enriched in MBP-TUT7 coIP.(F) Result of RNA coIP after in vivo UV-crosslinking with FLAG-TUT7 from control cells (transfected with control non-targeting siRNA, CNTRL) or cells depleted of MOV10 (by siRNA), both transfected with plasmids encoding L1 reporter and FLAG-TUT7. L1 enrichment was calculated by 2ˆΔΔCt method of L1 mRNAs enriched in each condition and normalized to GAPDH recovered in each condition.(E and F) Results of four independent biological experiments are shown. Statistical significance was calculated using a two-tailed Mann-Whitney test. Median values, with individual points and interquartile ranges are shown.See also Figure S5 and Tables S6 and S7.

Fig 3: 3′ RACE-Seq of L1 and Control mRNAs(A) Fraction of uridylated endogenous L1 mRNAs in human embryonic carcinoma cells (PA-1), human embryonic stem cells (H9-hESCs), human neuronal progenitor cells (derived from hESCs, NPC) and in mouse testes (of P10 young mice; 4 mice, 8 testes).(B) Distribution of 3′ tails in endogenous L1 mRNAs. The tails were assigned to one of four classes: U-tail (mono- and oligouridylated, but not adenylated); AU-tail (adenylated and mono- and oligouridylated); “no tail” (neither adenylated nor uridylated, mostly truncated within the 3′ UTR); A-tail (oligo- and polyadenylated).(C) Effect of siRNA-mediated depletion of MOV10 or TUT4 and TUT7 on uridylation of endogenous L1 mRNAs in PA-1. Statistical significance was calculated using one-way ANOVA and Tukey’s multiple comparison test. The comparison and significance are shown relative to a non-targeting siRNA control (CNTRL, ∗∗p < 0.01).(D) Uridylation of reporter L1 mRNAs in HEK293 cells under overexpression of MBP (CNTRL), WT, and MT TUT7, TUT4, or MOV10 as indicated. Statistical significance was calculated like in (C).(E) Distribution of 3′ tails in reporter L1 mRNAs, visualized like in (B) under MBP, TUT4, TUT7, or MOV10 overexpression conditions as indicated. White-dashed line and black-dashed line indicate control sample levels of uridylated (U+AU-tails) and adenylated L1 mRNAs, respectively.(F) Effects of overexpression of TUT1 and TENT5C on uridylation of reporter L1 mRNAs in HEK293 cells. Statistical significance was calculated like in (C).(G) Effects of depleting TUT4, TUT7, or both TUTases using siRNAs in HEK293 cells on uridylation of reporter L1 mRNAs. Statistical significance was calculated like in (C).(H) Distribution of 3′ tails in reporter L1 mRNAs, visualized like in (B) under TUT4, TUT4, and TUT7 or TUT7 depletion conditions in HEK293 cells as indicated.(I) Distribution of endogenous L1 and control mRNAs’ (ACTB, GAPDH, and SOGA2) 3′ ends in the cytoplasmic and nuclear compartments of PA-1 cells. The numbers of sequenced 3′ RACE reads are indicated and plotted assuming cyto+nuc = 100%. Qualities of the mRNAs’ 3′ ends are color-coded like in (B).Data in (A), (C), (D), (F), and (G) are medians with individual points and interquartile ranges shown. See also Figures S2 and S7 and Tables S1 and S2.

Fig 4: Stable Cell Line Validation and Co-IP Experiments, Related to Figure 5(A) Flow cytometry profiles of parental HEK293 FLP-IN T-Rex (black traces) and stable cell lines expressing EGFP-TUT4 or EGFP-TUT7 in the absence of (blue traces) or following induction of transgene expression with 100 ng/ml tetracycline (green traces). “EGFP+ GATE” denotes the region with cells showing higher EGFP fluorescence than ~99.9% of the control cells that do not express EGFP. The histograms were obtained using Flowing software. The table below the histograms summarizes the percentage of EGFP+ cells within each experimental population.(B) Western blot validation of the EGFP-TUT4-expressing stable cell line. Cells were grown for 48 h without tetracycline or with addition of 25, 50 or 100 ng/ml tetracycline in the medium. Proteins were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes and Ponceau S staining (lower panel) to control for protein loading. The upper panel shows results after probing with a TUT4-specific rabbit polyclonal antibody.(C) Western blot validation of the EGFP-TUT7-expressing stable cell line as in (B). Lanes 1 and 5 are reference lanes with material from the EGFP-TUT4 cell line and parental HEK293 FLP-IN T-Rex cells, respectively, to show antibody specificity. An asterisk denotes an unspecific band.(D) HEK293 FLP-In T-Rex cells were fixed with formaldehyde and stained for endogenous TUT4 (upper panel) or TUT7 (lower panel) with rabbit polyclonal antibodies and Alexa 488-coupled secondary goat anti-rabbit antibodies. Nuclei were visualized by Hoechst DNA staining (cyan). Maximal projection images of z stacks are shown. White bars, 10 µm(E) Single z-slides (epifluorescent – left, and bright field – right) of live cells from stable cell lines expressing either EGFP-TUT4 (upper panel) or EGFP-TUT7 (lower panel). White bars, 10 µm(F and G) Rapid cell fractionation following the protocol described by Suzuki et al. (2010) and subsequent western blotting to independently assess subcellular localization of TUT7, TUT4, MOV10. Blotting for cytoplasmic (tubulins or GAPDH) and nuclear (fibrillarin, RRP6 nuclear exosome complex exoribonuclease) markers were also performed. Cells were either parental HEK293 FLP-IN T-Rex (F), or HeLa-HA used in L1 mneoI reporter assays (G). An asterisk denotes an unspecific band.(H) Western blotting of proteins associated with either EGFP-MOV10 or EGFP showed are: blot for TUT7 probing (left), probing with monoclonal aEGFP antibody (middle; 1% co-IP), and polyclonal anti-TUT7 antibodies (right, 5% co-IP, blot before probing depicted on the left). Visible is TUT7 in EGFP-MOV10 co-IP and not in control EGFP co-IP. In TUT7-probed blot some cross-reactivity toward overrepresented EGFP-MOV10 but not EGFP can also be seen.(I) Flow-chart of the workflow of the RNA co-IP with FLAG-TUT7.(J) western blotting showing efficient depletion of MOV10 in HEK293T cells used for in vivo UV-crosslinking and co-IP with FLAG-TUT7. Shown are western blotting results after probing with polyclonal antibodies against MOV10 and GAPDH (loading control). Cells were transfected with: Lane 1 – control non-targeting siRNA then plasmids encoding L1 reporter and MBP-TUT7; lane 2 – MOV10 targeting siRNA then plasmids encoding L1 reporter and FLAG-TUT7; lane 3 – control non-targeting siRNA then plasmids encoding L1 reporter and FLAG-TUT7; lanes 4 and 5 – as in lane 3 but 0.5 and 0.2 of the material seen in lane 3 was loaded (control to compare with lane 2).(K) SDS-PAGE and silver staining of proteins recovered in the MBP- and FLAG-TUT7 co-IP after in vivo UV-crosslinking. Visible are bands representing FLAG-TUT7 (lanes 2 and 3, indicated with an asterisk) and M2 antibody stripped off the beads (lanes 2-4). Loaded were ca. 10% recovered material (lanes 2, 4) and ~6% recovered material (lane 3). Lane 1 – molecular weight ladder (170 and 55 kDa bands are indicated); lane 2 – IP with MBP-TUT7 (control); lane 3 – IP with FLAG-TUT7 from MOV10-depleted cells; lane 4 – IP with FLAG-TUT7 from control cells.The western blotting exposures were done either to a film and scanned by an Epson scanner and either top or bottom scanning options (panels B and F respectively) or by a CCD camera (C, G, H, J panels). The signals in the C, G, H and J panels were digitally enhanced by using ‘adjust levels’ option for the entire images (but for the middle H panel).

Fig 5: Control Experiments for Plasmid-Based L1 Retrotransposition Assays, Related to Figure 1(A) Western blotting to show expression of N’-MBP-tagged WT and MT TUT4, TUT7, MOV10 (lanes 1-5), N’-FLAG-tagged MT and WT TUT7 and MOV10 (lanes 6-8), C’-FLAG-tagged TENT4B, TENT2, TUT1 and TENT5C (lanes 9-12). Blots were probed with mouse monoclonal antibodies against MBP or rabbit polyclonal antibodies against FLAG. A probing for ?-tubulin and ponceau S staining were added as loading controls. A black arrow points to weakly expressed TENT2-FLAG in lane 10.(B and D) A plasmid encoding EGFP was used to test transfection efficiencies and toxicity (EGFP expression) concomitantly with co-transfection of a plasmid overexpressing wild-type or mutant TUT4, TUT7, MOV10 or MBP (CNTRL, B) or concomitantly with siRNA-directed depletion of both TUT4/7, TUT4, TUT7, MOV10 or non-targeting control (CNTRL, D). Data for 9 biological replicates (three independent experiments; panel B) and 3–6 biological replicates (two independent experiments, D) were normalized to controls. Means with SEM are plotted. No significant differences were observed as assessed by one-way ANOVA and Tukey’s multiple comparison test.(C) L1 retrotransposition assay in HEK293T cells with L1-megfpI reporters and concomitant overexpression of the indicated protein (as in panel A). Normalization was done to TUT7 MT. Statistical significance was calculated using one-way ANOVA and Tukey’s multiple comparison test. Statistical significance of TUT7 WT condition versus TUT7 mutant and the TENTs is shown (***p < 0.001).(E) Western blotting to test depletion of endogenous TUT4, TUT7, both TUTases or MOV10 by siRNAs (probed with specific antibodies; probing with a-tubulin was used as a loading control). Cells were co-transfected with the L1 megfpI reporter concomitantly with siRNAs. Cells were collected on day 4 post-transfection and split for flow-cytometry and western blotting. An asterisk marks an unspecific band detected by the anti-TUT7 antibodies (the band can be used to assess loading). Probing with the anti-a-tubulin mouse monoclonal antibodies showed 2 bands and was not used in other blots in the paper.(F) RT-qPCR estimation of TUT1 depletion at mRNA level by siRNAs at day 3 post-transfection (in cells co-transfected with the L1 megfpI reporter). Expression was normalized to control.(G) Western blotting to test depletion of TUT4 and TUT7, MOV10 or both TUTases and MOV10 in HeLa-HA cells under conditions used for retrotransposition assay with the mneoI reporter. Cells were collected at day 3 post-transfection (after co-transfection with L1-mneoI plasmids).Data on panels C and F are presented as medians with individual points and interquartile ranges shown.The western blotting exposures were done either to a film and scanned by an Epson scanner and bottom scanning option (panel G) or by a CCD camera (panels A and E). The singnals in the images acuired with a CCD camera were digitally enhanced by using ‘adjust levels’ option for the entire images.