Fig. 5: The HELZ2 helicase activity is critical for L1 inhibition. | Nature Communications

Fig. 5: The HELZ2 helicase activity is critical for L1 inhibition.

From: The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction

Fig. 5: The HELZ2 helicase activity is critical for L1 inhibition.The alternative text for this image may have been generated using AI.

a Schematic of the HELZ2 protein domains. HELZ2 contains two putative helicase domains (helicase 1 and helicase 2), which surround a putative RNB exonuclease domain. Open triangles, positions of missense mutations in conserved amino acids within the Walker A (WA) boxes in the helicase 1 and helicase 2 domains (K550A [WA1] and K2180A [WA2], respectively). Red arrowheads, relative positions of the 3xMYC carboxyl-terminal epitope tag in the HELZ2 expression constructs. b The effect of mutations in the Walker A box on L1 retrotransposition in HEK293T cells. HEK293T cells were co-transfected with cepB-gfp-L1.3, which contains an mEGFPI retrotransposition indicator cassette, and either pCMV-3Tag-8-Barr (control), pALAF015 (WT HELZ2), or one of the following HELZ2 expression plasmids that contain a mutation(s) in the Walker A box: pALAF025 (WA1); pALAF026 (WA2); or pALAF027 (WA1&2). Cells co-transfected with cepB-gfp-L1.3RT(-) intronless and either pCMV-3Tag-8-Barr, pALAF015 (WT HELZ2), or a mutant HELZ2 plasmid served as transfection normalization and toxicity controls. Top, timeline of the assay for experiments shown in panels (b) and (c). X-axis, name of HELZ2 expression constructs co-transfected into cells with cepB-gfp-L1.3; control, pCMV-3Tag-8-Barr. Y-axis, relative retrotransposition efficiency normalized to the cepB-gfp-L1.3 + pCMV-3Tag-8-Barr control. Pairwise comparisons relative to the cepB-gfp-L1.3 (mEGFPI) + pCMV-3Tag-8-Barr control: p = 2.5 × 10−11*** (WT HELZ2); 3.5 × 10−11*** (WA1); 1.7 × 10−6*** (WA2); and 0.070ns (WA1&2). c The effect of mutations in the Walker A box on L1 retrotransposition in HeLa-JVM cells. HeLa-JVM cells were co-transfected as in panel (b). Retrotransposition efficiencies were calculated as described in panel (b). Pairwise comparisons relative to the control: p = 0.00087*** (WT HELZ2); 0.26ns (WA1); 0.32ns (WA2); and 0.32ns (WA1&2). d Mutations in the HELZ2 helicase domains reduce the ability to inhibit L1 ORF1p and RNA. HeLa-JVM cells were transfected with pTMF3 (L1 ORF1p-T7), denoted by + symbol, and either pCMV-3Tag-8-Barr (control), pALAF015 (WT HELZ2), or an individual HELZ2 expression plasmid containing a mutation(s) in the Walker A box: pALAF025 (WA1), pALAF026 (WA2), or pALAF027 (WA1&2). Top: L1 RNA levels were determined by RT-qPCR using primers directed against sequences in the transfected L1 RNA (primer set: L1 [SV40]) and then were normalized to ACTB RNA levels (primer set: Beta-actin). Pairwise comparisons relative to the pTMF3 (L1 ORF1p-T7) + pCMV-3Tag-8-Barr control: p = 9.5 × 10−9*** (WT); 1.9 × 10−8*** (WA1); 7.3 × 10−7*** (WA2); and 1.5 × 10−6*** (WA1&2). Pairwise comparisons relative to the pTMF3 (L1 ORF1p-T7) + WT HELZ2: p = 0.56ns (WA1); 5.9 × 10−4*** (WA2); 1.9 × 10−4*** (WA1&2). Bottom: western blot image displaying ORF1p-T7 bands. HELZ2 expression was detected using an anti-MYC antibody. ORF1p was detected using an anti-T7 antibody. Pan-actin served as a sample processing control. e Small-interfering RNA (siRNA)-mediated knockdown of endogenous HELZ2 increases L1 retrotransposition. Top, timeline of the assay conducted in HeLa-JVM cells. Cells were transfected with a non-targeting siRNA control (siCtrl), siRNA targeting HELZ2 (siHELZ2), or siRNA targeting MOV10 (siMOV10). Middle left panel, HELZ2 RNA levels in siRNA treated cells. Middle right panel, MOV10 RNA levels in siRNA treated cells. X-axes, name of the siRNA. HELZ2 and MOV10 RNA levels were determined using RT-qPCR (primer sets: HELZ2 and MOV10, respectively) and then were normalized to ACTB RNA levels (primer set: Beta-actin). Y-axes, relative HELZ2 or MOV10 RNA levels normalized to the siCtrl. A two-tailed, unpaired Student’s t-test was used to calculate the p-values relative to the siRNA control: p = 3.1 × 10−5*** (siHELZ2); and 5.2 × 10−5*** (siMOV10). Bottom panel, HeLa-JVM cells were transfected with either siCtrl, siHELZ2, or siMOV10, followed by transfection with either cepB-gfp-L1.3 or cepB-gfp-L1.3RT(-) intronless, which was used to normalize transfection efficiencies. X-axis, name of the siRNA. Y-axis, relative retrotransposition efficiency. Pairwise comparisons relative to the non-targeting siRNA control: p = 2.9 × 10−4*** (siHELZ2); and 2.0 × 10−7*** (siMOV10). All the reported values represent the mean ± SEM from three independent biological replicates. The p-values, except for the RT-qPCR experiment shown in panel (e), were calculated using a one-way ANOVA followed by a Bonferroni–Holm post hoc tests. ns: not significant; * p < 0.05; *** p < 0.001.

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