Processing bodies are not required for mammalian nonsense-mediated mRNA decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.

FIGURE 1.

An ATPase-deficient UPF1 mutant, UPF2, and UPF3b localize to P-bodies in HeLa cells. (A) HeLa cells were stained with patient autoimmune serum IC6, which mainly contains α-Ge-1 antibodies, to visualize the P-bodies (shown in red; left column). HA-tagged UPF1 WT or the ATPase-deficient HA-UPF1 mut1 (Kashima et al. 2006) was expressed from transfected plasmids and detected with α-HA antibody (shown in yellow; middle column). The merge of both channels is shown in the right column. Endogenous (B) UPF2 and (C) UPF3b was visualized using rabbit polyclonal antisera against the respective proteins (Lykke-Andersen et al. 2000). Insets in the upper-left corner of the pictures in panels B and C show a single P-body zoomed in.

FIGURE 2.

The localization of HA-UPF1 mut1, UPF2, and UPF3b to cytoplasmic foci depends on the integrity of the P-bodies. (A) HeLa cells were transfected with a plasmid encoding a shRNA targeting Ge-1 or encoding a shRNA with no predicted target in human cells (control). The cells were stained with the IC6 serum (red) and α-DCP1a antibodies (green). (B) Ge-1 was knocked down, using the same conditions as in A, and HA-UPF1 WT or HA-UPF1 mut1 were co-transfected. The cells were stained with the IC6 serum (red) to visualize the P-bodies, and with α-HA antibody (yellow). (C,D) Ge-1 knockdown as in A and staining of the cells with the IC6 serum (red), with (C) α-UPF2 or (D) α-UPF3b antibodies (green).

FIGURE 3.

The disruption of P-bodies does not influence NMD. (A) Schematic depiction of the previously characterized NMD reporter transcript β-globin NS39 (Thermann et al. 1998). The open reading frame is shown as gray bar, exon–exon junctions are depicted by white lines, and the amino acid position of the PTC is indicated. (B–D) The efficacy of the RNAi-mediated Ge-1, UPF1, SMG1, and DCP2 knock-downs is monitored by Western blot analysis. Detection of SmB/B′ or Lamin A/C served as loading control. (E) A Ge-1 knockdown was performed as in Figure 2A, and additionally, the NMD reporter gene β-globin was co-transfected. The steady-state mRNA levels were quantified by RT-PCR and normalized to the miniμ WT mRNA from a co-transfected plasmid. (F) Relative mRNA levels of the three endogenous NMD substrates MAP3K14, SMG5, and PRKAB1 were measured after knockdown of Ge-1, UPF1, SMG1, or DCP2. Endogenous GAPDH mRNA levels were used for normalization. Average values from one representative experiment with three real-time PCR runs each are shown.

FIGURE 4.

The ATPase-deficient UPF1 localizes to P-bodies independent of SMG1, UPF2, and UPF3b. (A) The efficacy of the knockdowns was monitored by Western blot analysis. Detection of SmB/B′ served as loading control. (B) Western blot of SMG1-depleted cells expressing HA-UPF1 WT, HA-UPF1 mut1, or HA-UPF1 mut2. Detection of SmB/B′ served as loading control. (C) Cells expressing HA-UPF1 mut1 and with a knockdown of UPF2 (second row), UPF3b (third row), UPF2 and UPF3b together (fourth row), or with SMG1 (bottom row) were stained with the IC6 serum (red) and with α-HA antibodies (yellow). (D) HeLa cells transfected with HA-UPF1 mut2 were stained with the IC6 serum (red) and with α-HA antibodies (yellow).