HELZ directly interacts with CCR4–NOT and causes decay of bound mRNAs

(A) Schematic overview of the pentameric human CCR4–NOT complex used for in vitro interaction studies. The pentameric subcomplex is composed of NOT1 (residues E1093–E2371), CAF1, CAF40 (residues R19–E285), NOT2 (residues T344–F540), and NOT3 (residues L607–Q753). The CAF1 module contains the NOT1 MIF4G-like domain and CAF1 (green). The CAF40 module consists of CAF40 (blue; residues R19–E285) bound to the CAF40-binding coiled coil domain (CC; residues V1351–L1588). The adjacent NOT1 MIF4G-C (CD; residues D1607–S1815) is depicted in yellow. The NOT module consists of NOT1 (residues H1833–M2361), NOT2 (residues M350–F540; purple), and NOT3 (residues L607–E748; red). (B, C) In vitro MBP pull-down assay showing the interaction of recombinant MBP-Hs HELZ-C1-GB1-His (B) or MBP-Hs HELZ-C2-GB1-His (C) with distinct recombinant and purified CCR4–NOT modules (indicated on top of the respective gel). MBP served as a negative control. Input (33%) and eluted fractions (55%) were analysed by SDS–PAGE and Coomassie Blue staining.

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(A) Tethering assay in HEK293T cells using the β-globin-6xMS2bs reporter and MS2-HA–tagged HELZ (full-length or indicated fragments). The control reporter lacking the MS2bs (control) contains the β-globin gene fused to a fragment of the gapdh gene. The graph shows the quantification of mRNA levels of the β-globin-6xMS2bs reporter normalized to the levels of the control reporter and set to 100 for MS2-HA; the mean values ± SD are shown for four independent experiments. (B) Representative Northern blot of samples shown in (A). (C) Representative Western blot depicting the equivalent expression of the MS2-HA–tagged proteins used in (A) and (B). GFP served as a transfection control. (D) Immunoprecipitation assay in HEK293T cells showing the interaction of GFP-tagged HELZ wild-type (WT) and F1107V mutant with endogenous PABPC1. GFP-MBP was used as a negative control. Input (1.2%) and bound fractions (20% for GFP-tagged proteins and 35% for endogenous PABPC1) were analysed by Western blotting. (E) Tethering assay as described in (A), in cells expressing MS2-HA–tagged HELZ WT and F1107V mutant as indicated. The mean values ± SD are shown for four independent experiments. (F) Representative Northern blot of samples used in (E). (G) Western blot depicting the equivalent expression of the MS2-HA-HELZ WT and F110V in (E) and (F). GFP served as a transfection control. (H) Tethering assay as described in (A), but the transfection mixture included additionally plasmids expressing GFP-CAF1* and GFP-NOT1-Mid to block deadenylation (blue bars). GFP-MBP was overexpressed in control samples (black bars). The mean values ± SD are shown for three independent experiments. (I) Northern blot with representative RNA samples from the experiment depicted in (H). (J) Western blot showing the equivalent expression of HA-HELZ and the GFP-tagged proteins used in (H) and (I). Tubulin served as loading control. (K) Tethering assay as described in (A). The transfection mixture additionally included a plasmid expressing GFP-DCP2* catalytic mutant to block decapping (red bars). GFP was overexpressed in control samples (black bars). Tethering of MS2-HA-NOT1 served as positive control for deadenylation-dependent decapping (Kuzuoglu-Ozturk et al, 2016). The mean values ± SD are shown for three independent experiments. (L) Northern blot of representative RNA samples corresponding to the experiment shown in (K). The position of the fast migrating deadenylated form of the reporter mRNA (A₀) is marked with a red dotted line, whereas the position of the reporter with an intact poly(A) is indicated as (A_n). (M) Western blot showing the expression of HA-HELZ, HA-NOT1, and the GFP-tagged proteins used in (K) and (L). Tubulin served as loading control and V5-SBP-MBP as a transfection control. Transfection efficiency and/or plasmid expression was decreased in cells expressing GFP-DCP2*.

Source data are available for this figure.

(A–D) Immunoprecipitation assays in Dm S2 cells showing the interaction of GFP-Dm HELZ with HA-tagged-Dm HPat (A), HA-tagged-Dm PAN3 (B), HA-tagged-Dm NOT1 (C), and HA-tagged-Dm NOT2 (D). F-Luc-GFP served as negative control. Input (3.5% for GFP-tagged proteins and 0.5% for HA-tagged proteins) and bound fractions (10% for GFP-tagged proteins and 35% for HA-tagged proteins) were analysed by Western blotting. (E) Tethering assay in Dm S2 cells using the F-Luc-5BoxB reporter and λN-HA-Dm HELZ (full-length and fragments). A plasmid expressing R-Luc served as transfection control. F-Luc mRNA levels were normalized to those of the R-Luc control and set to 100 in cells expressing λN-HA. Graph shows the mean values ± SD of four experiments. (F) Representative Northern blot of samples shown in (E). (G) Western blot showing the equivalent expression of the λN-HA–tagged proteins used in (E). GFP-V5 was used as transfection control. (H) Dm S2 cells were treated with dsRNA targeting glutathione S-transferase (control) or DCP1 and Ge-1 mRNAs. The efficacy of the KD was estimated by Western blot with antibodies specifically recognizing endogenous DCP1 and Ge-1 proteins. PABP served as a loading control. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of the depletion. The asterisks (*) mark unspecific bands recognized by the respective antibody. (I, J) Dm S2 cells treated with dsRNA targeting either glutathione S-transferase (control, green bars) or DCP1 and Ge-1 mRNAs (yellow bars) were transfected as described in (E). Tethering of λN-HA-GW182 served as positive control for deadenylation-dependent decapping (Behm-Ansmant et al, 2006). Panel (I) shows relative F-Luc activity in control and DCP1 + Ge-1 KD samples. Panel (J) depicts relative F-Luc mRNA levels in control and DCP1 + Ge-1 KD samples. The mean values ± SD are shown for five independent experiments. (K) Representative Northern blot analysis of samples shown in (J). The position of the fast migrating deadenylated form of the reporter mRNA (A₀) is marked with a red dotted line, whereas the position of the reporter mRNA with intact poly(A) is indicated as (A_n). (L) Western blot showing the equivalent expression of the λN-HA–tagged proteins used in (I). F-Luc-V5 was used as transfection control.

Source data are available for this figure.

(A, B) Tethering assay in HEK293T cells using the R-Luc-6xMS2bs-A₉₅-MALAT1 reporter with MS2-HA-HELZ WT and F1107V mutant. A plasmid coding for F-Luc-GFP served as control. Shown is the quantification of protein (A) and of mRNA levels (B) of the R-Luc-6xMS2bs-A₉₅-MALAT1 reporter normalized to the levels of the control reporter and set to 100 for MS2-HA. The mean values ± SD are shown for four independent experiments. (C) Representative Northern blots of samples shown in (B). (D) Western blot showing the equivalent expression of the MS2-HA tagged proteins used in (A). F-Luc-GFP was used as transfection control. (E) Western blot analysis of HeLa cells after NOT1 KD. Dilutions of control cell lysates were loaded in lanes 1–4 to estimate the efficacy of NOT1 depletion. Transfected MS2-HA-HELZ protein was expressed at comparable levels in WT and NOT1 KD cells. PABPC1 served as a loading control. (F) Tethering assay in HeLa cells using the R-Luc-6xMS2bs-A₉₅-MALAT1 reporter and MS2-HA-HELZ. HeLa cells were treated with scrambled shRNA (green bar) or shRNA targeting NOT1 mRNA (grey bar). The graph shows relative R-Luc activity in control and NOT1 KD samples. The mean values ± SD are shown for three independent experiments. (G) Tethering assay in HEK293T WT (green bars) and Ddx6-null cells (blue bars) with MS2-HA-HELZ and the R-Luc-6xMS2bs-A₉₅-MALAT1 reporter. For complementation studies, the cells were also transfected with either GFP or GFP-DDX6. A plasmid expressing F-Luc-GFP served as a transfection control. Shown is the quantification of R-Luc activity normalized to F-Luc activity and set to 100 for MS2-HA in WT or Ddx6-null cells. The mean values ± SD are shown for three independent experiments. (H) Western blot showing the levels of transfected MS2-HA-HELZ protein in the different cell lines used in (G). Loss of endogenous DDX6 protein expression in HEK293T Ddx6-null cells was confirmed using an anti-DDX6 antibody (lane 2, middle panel). The blot further illustrates that GFP-DDX6 was expressed at a level equivalent to endogenous DDX6 (lane 3 versus lane 1). F-Luc-GFP served as transfection control.

Source data are available for this figure.

(A) Pie chart indicating the fractions and absolute numbers of differentially expressed genes derived from the analysis of the transcriptome of HEK293T wild-type (WT) and Helz-null cells by RNA-Seq. Two biological replicates of each cell line were analysed. The RNA-Seq analysis indicated that 7,466 (grey) of the total 10,978 genes selected using fragments per kilobase of transcript per million mapped reads >2 cut-off showed no significant differences between the two cell lines (FDR ≥ 0.005). 1,682 genes were significantly up-regulated (red) whilst 1,830 genes were down-regulated (blue) using an fold change (FC) >0 on log₂ scale with an FDR < 0.005 to determine abundance. (B) Gene ontology analysis of the biological processes overrepresented in the group of transcripts up-regulated in Helz-null cells (log₂FC > 0, FDR < 0.005) versus all other expressed genes. Bar graph shows −log₁₀ of q values for each category. Content of brackets indicates the number of genes within each category. (C) Western blot analysis depicting the levels of endogenous HELZ present in HEK293T WT cells (lane 1) compared with Helz-null cells transfected with either 1 or 4 μg of GFP-HELZ (lanes 2 and 3, respectively). Tubulin served as loading control. (D) qPCR validation of three up-regulated (log₂FC > 0, FDR < 0.005) transcripts identified in (A). Transcript levels of sparc (blue bars), basp1 (orange bars) and tenm1 (grey bars) were determined in HEK293T WT, Helz-null, and Helz-null cells complemented with either 1 or 4 μg of GFP-HELZ. Transcript levels were normalized to gapdh mRNA. Shown are the normalized expression ratios ± SD for three independent experiments.