A translational silencing function of MCPIP1/Regnase-1 specified by the target site context

Figures

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  Figure 1.. Co-precipitation of mRNAs with MCPIP1. (A) Lysates of HeLa cells expressing GFP-tagged RNase-deficient MCPIP1 (GFP-MCPIP1(D141N)) (G-M) or GFP (G) as a control were analyzed by western blots with antibodies against MCPIP1 (upper panel). Aliquots from lysates (input) and from beads after pulldown (PD) were analyzed by antibodies against GFP (lower panel) (asterisk: heavy and light chains of anti-GFP antibodies applied for pulldown). (B and C) Total RNA was isolated from lysates and from beads after pulldown and mRNAs were quantitated by RT-qPCR. In (C) plasmids expressing β-globin mRNA without (βG) or with the NFKBIZ TSE inserted into the 3′ UTR (βG-TSE) were co-transfected. Results are expressed as fold increase in the ratio of pulldown over input for GFP-MCPIP1(D141N) as compared to GFP (set as 1) (mean ± SD for one out of three experiments with similar results; asterisk: P < 0.01 for GFP-MCPIP1(D141N) > GFP).

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  Figure 2.. Increased TSE-mediated translational silencing in cells overexpressing MCPIP1. (A) HEK-293 cells were co-transfected with a GFP expression plasmid containing the TSE in its 3′ UTR (GFP-TSE) without (con) or with strep-tagged MCPIP1. Cell lysates were subjected to western blot in serial dilutions using antibodies against GFP or eEF2 as a loading control. (B) GFP-TSE mRNA was quantified by RT-qPCR (mean ± SD of quadruplicates from one out of three experiments with similar results). (C) HEK-293 cells were transfected with plasmids encoding GFP mRNAs without (GFP) or with the NFKBIZ TSE (GFP-TSE) together with empty vector (con) or strep-tagged MCPIP1 as indicated. Fluorescence intensity was determined by flow cytometry. (D) Mean fluorescence intensities (MFI,±SD of triplicates; n.s., not significant) of cells transfected with plasmids encoding GFP-TSE together with empty vector (con) or strep-tagged WT or RNase-deficient MCPIP1 (D141N). (E) Fold suppression of protein (determined as MFI) and mRNA amounts (determined by RT-qPCR and normalized to GAPDH mRNA) by the TSE or by strep-MCPIP1 expression or both was calculated by setting the values for GFP mRNA without TSE and in the…

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  Figure 3.. Impaired TSE-mediated translational silencing in MCPIP1-depleted cells. (A) Western blot detection of endogenous MCPIP1 in HeLa cells expressing Cas9 nuclease without or with two different sgRNAs specific for the MCPIP1-encoding ZC3H12A gene (sgZ #2 and sgZ #4), and of the two HeLa cell clones established from bulk cultures expressing sgZ #2. (B) HeLa cells analyzed in (A) were transfected with firefly luciferase plasmids without (−) or with the TSE. Relative luciferase activity was calculated as the ratio of firefly- to co-expressed Renilla luciferase-generated light units. Fold suppression (mean ± SD) by the TSE represents the ratio of activity for plasmids without TSE (set as 1) over activity for TSE-containing plasmid. (C) In HeLa cells expressing Cas9 without or with the indicated sgRNA, firefly luciferase activity generated by plasmids without (−) or with the TSE was quantified and normalized to the amounts of the corresponding mRNA. Shown is TSE-mediated fold suppression of normalized luciferase activity (mean ± SD from one out of three assays with similar results). TSE-mediated suppression of mRNA amounts was 1.9-, 1.0- and 1.6-fold in WT, sgZ #2 and sgZ #4 cells,…

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  Figure 4.. Involvement of TSE substructures in MCPIP1 binding and function. (A) Mammalian sequences matching the TSE were identified using BLAST. A combined sequence structure alignment was obtained using mLocarna followed by RNAalifold. The number of different types of base pairs for a consensus pair, i.e. compensatory mutations supporting the structure, is given by the hue, the number of incompatible pairs by the saturation of the consensus base pair. (B) Scheme of TSE and reporter mRNAs expressed and summary of results obtained for them ((13), this report and additional data, not shown). (C) HeLa cells were co-transfected with GFP-MCPIP1(D141N) and β-globin reporter constructs containing the indicated insertions. Co-pulldown of reporter mRNA with GFP-MCPIP1(D141N) was determined as in Figure 1. (D) Degradation kinetics of reporter mRNAs in HeLa cells were determined by quantification of residual mRNA at the indicated time points after blocking transcription with actinomycin D (Act. D, 5 μg/ml). (E) Degradation kinetics were determined as in (D) for HEK-293 cells expressing the indicated reporter mRNA without (con) or with strep-MCPIP1 (one out of three independent assays with similar…

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  Figure 5.. Domains of MCPIP1 involved in translational silencing. (A) Empty vector (con) or plasmids encoding WT and different mutants of strep-MCPIP1 (see scheme) were transfected into HEK-293 cells together with firefly luciferase plasmid without (−) or with the TSE inserted. Equal expression of MCPIP1 and mutants was controlled by western blot. (B) Firefly luciferase activity was normalized to the corresponding mRNA amount quantified by RT-qPCR. Results are expressed as fold suppression with the value for firefly luciferase activity per mRNA in the absence of the TSE and co-transfected MCPIP1 set as 1 (mean ± SD for one out of three assays with similar results; asterisk, P < 0.01 for WT versus mutant; n.s., not significant) (mutants: RNase/DUBdef, RNase and deubiquitination deficient; RNasedef, RNase deficient; DUBdef, deubiquitination deficient; Dimdef, dimerization deficient; ΔZF, zinc finger deleted; ΔPRR, proline-rich region deleted). (C) Binding of the indicated MCPIP1 mutants to the reporter mRNA was determined as in Figure 1, using StrepTactin beads for pulldown.

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  Figure 6.. Ribosome association of TSE-containing mRNAs. (A) Total RNA from HeLa cells transfected with plasmids expressing β-globin mRNA without (βG) or with the TSE (βG-TSE) was subjected to pulldowns with antibodies recognizing the cap structure or control antibodies. In vitro-transcribed luciferase RNA was used as a non-capped control RNA (cap(−)). Results (mean ± SD, n = 3) are expressed as fold increase in the ratio of pulldown over input for immunoprecipitation with specific as compared to control antibodies; n.s., not significantly enriched by cap-specific antibodies. (B) HeLa cells were transfected with plasmids encoding β-globin mRNA without (βG) or with the TSE (βG-TSE). RNA was isolated from pooled subpolysomal (SP) or polysomal (P) fractions after sucrose density centrifugation and analyzed by northern blot with a β-globin antisense probe. In vitro-transcribed β-globin-TSE RNA without poly(A)-tail (i.t. βG-TSE) was loaded as a size marker. The positions of βG-TSE mRNA (*), βG mRNA (**) and18 S rRNA are indicated, ethidium bromide staining of 28 S rRNA is shown for comparison of RNA amounts loaded. (C) Polysome profiles of the indicated mRNAs were determined for HeLa cells as in…

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  Figure 7.. Suppressive effect of the TSE and MCPIP1 on IRES-dependent translation. (A) HeLa cells were transfected with conventional firefly luciferase plasmids without (−) or with the TSE for cap-dependent translation or with the corresponding plasmids encoding a hairpin structure followed by the CrPV IRES and a mutated luciferase start codon for IRES-dependent translation (see scheme and Supplementary Figure S6A). Luciferase activity was normalized to luciferase mRNA amounts. Results are expressed as fold inhibition by the TSE as compared to luciferase without TSE (mean ± SD for one out of five assays with similar results). (B) In HEK-293 cells co-transfected with empty vector (con) or strep-MCPIP1 expression plasmid suppression of firefly luciferase activity by the TSE was determined for CrPV-dependent translation as in (A).