Fig 2: CCR4-NOT complex associates with LC3B, promoting efficient LMD.a IPs of endogenous LC3B. The extracts of HEK293T cells either treated or not treated with Rapa + CQ were subjected to IPs using a-LC3B antibody or nonspecific rIgG. The intensities of each western blot image were quantitated. The intensities of coimmunoprecipitated proteins were normalized to those of immunoprecipitated LC3B. The relative levels obtained in untreated cells were arbitrarily set to 1.0. n = 3. b, c IPs of endogenous LC3B upon downregulation of both ATG5 and ATG7. As performed in a, except that the cells were either undepleted or depleted of ATG5 and ATG7. b Western blotting proving specific downregulation of endogenous ATG5 and ATG7. c Western blotting of cellular proteins before or after IPs of LC3B. d The proximity ligation assay (PLA) between endogenous LC3B and either CNOT1 or CNOT7. PLA experiments using the indicated antibodies were performed on HeLa cells treated with either DMSO or Rapa + CQ. The PLA images are shown in Supplementary Fig. 10. The number of PLA spots per cell was quantified and is presented in this panel. Box-whiskers show maximum, third quartile to first quartile, median and minimum; n = 767 cells examined over three independent experiments. e, f Effect of CNOT1 or CNOT7 downregulation on LMD. e Western blotting showing specific downregulation of endogenous CNOT1 and CNOT7. f LMD efficiency of RLuc-P3'-WT reporter mRNA, endogenous PRMT1 mRNA, and MARS1 mRNA. n = 3; Data are presented as mean values ± SD; p values were analyzed using two-tailed and equal-variance Student’s t test; *p < 0.05; **p < 0.01 (The exact p values are provided in Source Data file). Source data are provided as a Source Data file.

Fig 3: Validation of interactome data by co-IP.(A) Co-IPs validating that UL72 interacts with CCR4-NOT Transcription Complex Subunits 7 and 2 (CNOT7 and CNOT2), conducted in HEK293T cells. For all experiments in this figure, left panels show an IB of 1–2% of input sample, and right panels shown an anti-V5 co-IP. Cells were transiently transfected with two plasmids, one expressing the C-terminally V5-tagged viral protein and the other expressing the C-terminally HA-tagged cellular prey. Bait proteins were detected with anti-V5, and prey with antibodies against CNOT7 or CNOT2 protein. Controls included GFP or the viral UL34 protein. CANX – calnexin loading control. This figure is representative of n = 1 experiment (CNOT2); n = 2 experiments (CNOT7). Expected sizes: CNOT7: 33 kDa; CNOT2: 52 kDa; CANX: 72 kDa; UL72: 44 kDa; UL34: 45 kDa. (B) Co-IPs validating that UL72 interacts with CNOT7 and CNOT2, conducted in HFFF-TERT cells overexpressing C-terminally V5-tagged UL72. Proteins were detected as described in (A). This figure is representative of n = 2 experiments (CNOT2); n = 1 experiment (CNOT7). Expected sizes: CNOT7: 33 kDa; CNOT2: 52 kDa; CANX: 72 kDa; UL72: 44 kDa; UL34: 45 kDa. (C) Co-IP validating the interaction between RL1 and CUL4A, conducted in HEK293T cells as described in (A), but with detection of CUL4A using anti-HA. This figure is representative of n = 4 experiments. Expected sizes: CUL4A: 77 kDa; RL1: 35 kDa; UL34: 45 kDa; CANX: 72 kDa. (D) HCMV UL71 interacted with multiple interferon-stimulated proteins, including TRIM22. (E) Co-IP validating the interaction between UL71 and TRIM22, conducted as described in (C). This figure is representative of n = 3 experiments. Expected sizes: TRIM22: 56 kDa; UL71: 40 kDa; UL34: 45 kDa; CANX: 72 kDa.

Fig 4: RNF219–CCR4–NOT exhibits deadenylation activity. (A) FLAG purification of RNF219 from the HEK-293 FIT cells stably expressing FLAG-tagged RNF219 and CNOT1 from the FLAG-CNOT1-expressing HEK-293 FIT cells. Levels of RNF219, CNOT1, and CNOT7 in the purified immuneprecipitates were examined by western blotting. (B) In vitro deadenylation of the synthesized N20A20 RNA using the affinity-purified FLAG-RNF219 or FLAG-CNOT1-containing immunoprecipitate. Samples were harvested at the indicated time intervals and resolved by electrophoresis separation. FLAG purification from HEK-293 FIT cell lysate was used as a negative control.

Fig 5: LBs are enriched with DDX6 and CNOT7 proteins.(A) Double immunofluorescence staining of RNA helicase DDX6 (green) and L1 ORF1p (magenta) in Mael+/- and Mael-/- testes. DDX6 is detectable in cytoplasmic foci in both Mael+/- and Mael-/- spermatogonia and spermatocytes (yellow arrows); in Mael-/- both ORF1p and DDX6 concentrate in LBs (white arrowheads). Dotted white lines mark seminiferous tubule boundaries. Scale bars: 10 µm. (B) Double immunofluorescence staining of CCR4-NOT complex subunit CNOT7 (green) and L1 ORF1p (magenta) in Mael+/+ and Mael-/- testes. CNOT7 is detectable in cytoplasmic foci in Mael+/+ spermatocytes (yellow arrows); in Mael-/- CNOT7 colocalizes with ORF1p in LBs (white arrowheads). Scale bars: 10 µm. (C) Double immunofluorescence staining of DCP1A (green) and L1 ORF1p (magenta) in Mael+/- and Mael-/- testes. DCP1A shows a characteristic granular pattern in both samples (yellow arrows), although PBs appear overall less abundant in Mael-/- seminiferous tubules; weak diffuse DCP1A signal is also detected in LBs (white arrowheads). Boxed areas in Mael-/- panels are magnified in insets. Scale bars: 10 µm. See also S6 Fig.