SUZ domain-containing proteins have multiple effects on nonsense-mediated decay target transcripts

Abstract

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.

Keywords: SUZ domain; SUZ-C domain; nonsense-mediated decay; protein domain; translation.

Figure 1

SZRD1 isoforms interact with UPF1 and STRAP.A, schematic representation of the SZRD1 gene locus and the resulting transcripts. Inclusion or exclusion of exon 2 (in yellow) lead to long and short isoforms, respectively. B, schematic representation of SZRD1 transcripts and the resulting proteins. C, Western blot analysis with antibodies predicted to recognize short and long SZRD1 isoforms as well as β-actin was performed in HEK293 cells expressing shRNAs specifically targeting the long (exon 2; “#3,” “#4,” “#5,” and “#6”) or all SZRD1 isoforms (exon 4, “#1” and “#2”). Samples labeled with “control” express a nonsilencing control shRNA. D, lysates from HEK293 cells overexpressing the indicated SZRD1 proteins (“SZRD1”) with a C-terminal SFB tag or an empty vector (“control”) were subjected to affinity purification and analyzed by Western blot using the indicated antibodies. Input samples correspond to 10% of the amount used in the pulldown. Exposure time was identical for input and pulldown samples except for Western blots analysing STRAP, where pulldown exposure was five times shorter (∗). E, lysates from HEK293 cells overexpressing SZRD1 protein isoforms (containing the additional arginine or lysine residue) with a C-terminal SFB tag identical to D were subjected to affinity purification in the presence (“+”) or the absence (“−”) of RNAse A/T1. Western blot analysis and presentation are identical to D. F and G, lysates from HEK293 cells overexpressing UPF1 with an N-terminal SFB tag (“UPF1”) (F), STRAP with an N-terminal SFB tag (“STRAP”) (G) or an empty vector (“control”) were subjected to affinity purification followed by Western blotting as described in D. H, schematic representation of the selection cassette used to insert an SFB tag before the stop codon of the endogenous coding sequence of UPF1. I, protein lysates from HEK293 cells carrying a C-terminal SFB tag knocked into the endogenous loci of UPF1 or an unmodified control cell line (“ctrl”) were subjected to affinity purification followed by Western blot analysis with the indicated antibodies. Input corresponds to 10% of the amount used in the pulldown. Exposure time was identical for input and pulldown samples except for Western blots analyzing UPF1, where pulldown exposure was five times shorter (∗). HEK293, human embryonic kidney 293 cell line; STRAP, serine/threonine kinase receptor–associated protein; SZRD1, SUZ domain–containing protein 1; UPF1, up-frameshift 1.

Figure 2

The SUZ domain is required for the interaction with UPF1, and the SUZ-C domain is required for the interaction with STRAP.A and B, alignment of human SUZ-C domain (A) and SUZ domain (B) sequences of the indicated proteins (top panel) and graphical representation of the conservation of these sequences across evolution (humans, mouse, chicken, and frog) (lower panel). Mutations and deletions (“Δ”) used in the other panels are indicated. C and D, lysates from HEK293 cells overexpressing the C-terminally SFB-tagged SZRD1 long isoform (carrying the mutations illustrated in A and B) were subjected to affinity purification with streptavidin beads followed by Western blot analysis. Input corresponds to 10% of the amount used in the pulldown. Cell lines labeled with “control” were transduced with an empty control vector. Exposure time was identical for input and pulldown samples except for Western blots analyzing STRAP, where pulldown exposure was five times shorter (∗). Pulldown efficiency in D corresponds to the signal obtained in the pulldown relative to the signal obtained in the input sample. To facilitate comparisons, pulldown efficiency was normalized to the wildtype construct. E, schematic representation of SZRD1 interacting with UPF1 and STRAP. F and G, HEK293 cells expressing STRAP (H) or UPF1 (I) with an N-terminal SFB tag or empty vector control cells (“ctrl”) were engineered to express an shRNA targeting SZRD1 (“+”) or a nonsilencing shRNA (“−”). Protein lysates were subjected to affinity purification followed by Western blot analysis. Input corresponds to 10% of the amount used in the pulldown. HEK293, human embryonic kidney 293 cell line; STRAP, serine/threonine kinase receptor–associated protein; SZRD1, SUZ domain–containing protein 1; UPF1, up-frameshift 1.

Figure 3

Recruitment of SZRD1 increases protein production from NMD-sensitive reporter constructs.A, schematic representation of Renilla reporter gene constructs that allow recruitment of λN-tagged proteins. Boxes represent the ORFs, which contains the Renilla ORF (in white) followed by a genomic β-globin sequence (in brown). The “V” shape illustrates intron positions. In the 3’UTR, six BoxB sites (“BoxB”) and an AU-rich element (“ARE”) or a control sequence (“AREctrl”) were inserted. Locations of the primers used in the RT–qPCR are indicated in cyan, and the premature stop codon is indicated with “STOP”. A constitutively active firefly construct with the same promoter was used as transfection control. B and C, relative luciferase mRNAs (B, assessed by RT–qPCR) and activities (C, assessed by luminescent assay) in HEK293T cells after transfection of the indicated reporter gene constructs in the presence (“λN-SZRD1”) or the absence (“control” = empty vector) of plasmids driving expression of the long form of SZRD1 with a λN tag in the N terminus. Values represent the ratio of Renilla to firefly luciferase activities or mRNA levels and are means ± SD of three independent experiments, where each condition was performed in triplicates. Asterisks indicate p < 0.05 in post hoc testing after two-way ANOVA; ns denotes not significant. D, schematic representation of Renilla luciferase reporter gene constructs used in (E) that allow (“WT-BoxB” and “NMD-BoxB”) recruitment of λN-tagged SZRD1 via six BoxB sites or not (“WT-noBoxB” and “NMD-noBoxB”). Each construct contains the Renilla luciferase ORF (in white) followed by a genomic β-globin sequence (in brown) containing two introns. In the 3′UTR, six BoxB sites (“BoxB”) or not (“BoxB-ctrl”) were inserted. A premature stop codon (“STOP”) in the NMD constructs triggers NMD. E, relative luciferase activities observed for the reporter gene constructs (“WT” and “NMD”) with 6× BoxB sites (+) or not (−), when cotransfected in the presence (gray bars) or the absence (black bars, “ctrl” = empty vector) of plasmid driving expression of the λN-tagged long form of SZRD1 (“λN-SZRD1”). Values represent the ratios of Renilla to firefly luciferase activities and are means ± SD of three independent experiments, where each condition was transfected in triplicates. Asterisks indicate p < 0.05 in post hoc testing after two-way ANOVA; ns denotes not significant. HEK293T, human embryonic kidney 293 cell line; NMD, nonsense-mediated decay; qPCR, quantitative PCR; SZRD1, SUZ domain–containing protein 1.

Figure 4

SZRD1 knockdown decreases protein production from the NMD-sensitive mRNAs GABARAPL1 and PEA15.A, Western blot analysis using the indicated antibodies in HEK293 cell lines expressing a nonsilencing shRNA (“control”), two different shRNAs targeting SZRD1 (“1” and “2”), or one shRNA targeting UPF1. B and C, quantitative analysis of Western blots for GABARAPL1 normalized to GAPDH (B) and qPCR analysis for GABARAPL1 mRNA levels normalized to TBP and β2-microglobin (C) in cell lines described in A. Data represent means ± SD from three independent experiments and were normalized to the control shRNA condition. D, Western blot analysis of U2OS cell lines expressing a nonsilencing shRNA (“control”), two different shRNAs targeting SZRD1 (“1” and “2”), or one shRNA targeting UPF1. E–H, quantification of GABARAPL1 (E and F), PEA15 (G and H) protein levels (E and G), and mRNA levels (F and H) in U2OS cell lines were determined and are presented as shown in B and C. Post hoc testing after one-way ANOVA was performed in comparison to the control condition. Asterisks indicate comparisons with p < 0.05; not significant comparisons are not indicated. Blots presented in A are one of three experiments that were also used to generate Figure 7, A and F. As such, panels for SZRD1 and GAPDH in A and D are identical to the ones in Figure 7, A and F. HEK293, human embryonic kidney 293 cell line; NMD, nonsense-mediated decay; qPCR, quantitative PCR; SZRD1, SUZ domain–containing protein 1; UPF, up-frameshift.

Figure 5

The SUZ domain of SZRD1 and ARPP21 is required to increase protein production from NMD-sensitive reporter constructs.A, schematic representation of Renilla reporter gene constructs used in (B) and Fig. S3A. B, relative luciferase activities of the NMD-BoxB construct in the presence or the absence of λN-tagged long SZRD1 containing or not mutations in the SUZ and SUZ-C domains (Fig. 2, A and B). Values represent the ratio of Renilla to firefly luciferase activities and are means ± SD of three independent experiments, where each condition was performed in triplicates. Asterisks indicate p < 0.05 in post hoc testing after two-way ANOVA. C, hypothesis about the role of the SUZ domain of SZRD1 and ARPP21. D, HEK293 cells with an SFB tag knocked in the UPF1 locus or a control cell line (“control”) were transiently transfected with wildtype ARPP21, a deletion mutant lacking part of the SUZ domain, or an empty vector control (“control”). Protein lysates were subjected to affinity purification followed by Western blot analysis with the indicated antibodies. E and F, relative mRNA levels (E) and luciferase activities (F) obtained and presented as in panels of the Figure 3, B and C, except that expression constructs for N-terminally λN-tagged ARPP21 (gray bars) or a splice variant of ARPP21 missing a part of the SUZ domain (cyan bars) were transfected. HEK293, human embryonic kidney 293 cell line; NMD, nonsense-mediated decay; SZRD1, SUZ domain–containing protein 1.

Figure 6

Strong inhibition of NMD upon supraphysiological expression of SZRD1 likely does not represent the physiological function of SZRD1.A, RNA-Seq was performed 48 h after infection of HeLa cells with lentiviruses driving the overexpression of the long form of SZRD1 or an empty expression cassette. B, Western blot analysis of the infected HeLa cell lines expressing SZRD1 or not. C, GSEA with a gene set of NMD targets revealed strong enrichment among transcripts increased upon SZRD1 overexpression. Genes are sorted according to the signed t-statistic. Upregulated genes are on the left. D, volcano plot of gene expression changes upon overexpression of SZRD1. Members of the NMD target gene set are highlighted in red. E–H, mRNA levels of hnRNPA2B1 (I), ZFAS1 (J), SC-35 (K), and GAS5 (L) were measured by RT–qPCR in HEK cells expressing the indicating SZRD1 protein (“long” or “short”) or not (“ctrl” = empty vector control). Expression levels were normalized to TBP and β2-microglobin mRNA levels. Data represent means ± SD from three independent experiments and were normalized to the empty vector condition. I–L, mRNA levels of hnRNPA2B1 (I), ZFAS1 (J), SC-35 (K), and GAS5 (L) were measured by RT–qPCR in HEK cells expressing a nonsilencing control shRNA (“ctrl”), two shRNAs (“1” and “2”) targeting SZRD1, or one shRNA targeting UPF1. Expression levels were normalized to TBP and β2-microglobin mRNA levels. Data represent means ± SD from three independent experiments and were normalized to the control shRNA condition. FDR, false discovery rate; GSEA, gene set enrichment analysis; HEK, human embryonic kidney 293 cell line; NMD, nonsense-mediated decay; qPCR, quantitative PCR; SZRD1, SUZ domain–containing protein 1; UPF, up-frameshift.

Figure 7

Endogenous SZRD1 is required to maintain UPF3B proteins levels but only has minor effects on mRNA levels of NMD targets.A, Western blot analysis using the indicated antibodies in HEK293 cell lines expressing a nonsilencing shRNA (“control”), two different shRNAs targeting SZRD1 (“1” and “2”), or one shRNA targeting UPF1. B–E, quantification of Western blot signals with the indicated antibodies obtained in three independent experiments using HEK293 cell lines expressing a nonsilencing shRNA (“control”), two different shRNAs targeting SZRD1 (“1” and “2”), or one shRNA targeting UPF1. Signals were normalized to the abundance of GAPDH within each experiment and represent means ± SD. Post hoc testing after one-way ANOVA was performed in comparison to the control condition. Asterisks indicate p < 0.05; not significant comparisons are not indicated. F, Western blot analysis using the indicated antibodies in U2OS cell lines generated as described in A. G–J, quantification of Western blot signals with the indicated antibodies obtained in three independent experiments using U2OS cell lines as described for the B–E. K, experimental setup for the analysis of HeLa cells with doxycycline-inducible expression of an SZRD1 shRNA or a control shRNA. L, Western blot analysis demonstrating knockdown of SZRD1. M, GSEA revealed the enrichment of a gene set of NMD targets (7) among transcripts increased upon SZRD1 knockdown. Genes are sorted according to the signed t-statistic. Upregulated genes are on the left. N, volcano plot of gene expression changes upon SZRD1 knockdown. Members of the NMD target gene set are highlighted in red. O, schematic representation of the potential roles of SZRD1 based on our observations. FDR, false discovery rate; GSEA, gene set enrichment analysis; HEK293, human embryonic kidney 293 cell line; NMD, nonsense-mediated decay; SZRD1, SUZ domain–containing protein 1; UPF, up-frameshift.