Nudt3 is an mRNA decapping enzyme that modulates cell migration

Abstract

Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

Keywords: Nudt3; cell motility; fibronectin; integrin β6; mRNA decapping; mRNA stability.

FIGURE 1.

Nudt3 down-regulation increases expression of mRNAs implicated in cell motility. (A) RNA levels of nine mRNAs implicated in cell migration tested in MCF-7 cells expressing nontargeting shRNA (Con^KD, open bar), shRNA against Nudt3 (Nudt3^KD, filled bar), or shRNA against Nudt4 (Nudt4^KD, gray bar) are shown. (B) Western blot analysis of Nudt3 protein levels in Con^KD or Nudt3^KD MCF-7 cells ectopically expressing GFP, Flag-tagged wild-type Nudt3 (Nudt3), or mutant Nudt3 (Nudt3^EE/QQ). The band for Flag-Nudt3^EE/QQ runs slightly faster than that for Flag-Nudt3 likely due to differences in protein charge by the introduction of glutamic acid residues into the mutant protein. (C) A subset of the mRNAs implicated in cellular movement derived from the 144 mRNAs detected to increase greater than or equal to twofold by RNA-seq analysis in Nudt3^KD are shown. (D) Transcript levels of the indicated mRNAs were determined by qRT-PCR in Con^KD or Nudt3^KD cells complemented with the indicated proteins. RNA levels in A and D are derived from at least three independent experiments and normalized to 18S rRNA and presented relative to the value in control cells set to one. Error bars represent ±SD. P-values are denoted by asterisks. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 (Student's t-test).

FIGURE 2.

Nudt3 is a decapping enzyme that modulates stability of integrin β6 and lipocalin-2 mRNAs. Stability of the indicated mRNAs was determined following actinomycin D-mediated transcriptional silencing in Con^KD and Nudt3^KD cells complemented with the indicated proteins. Values are normalized to levels of 18S rRNA and presented relative to their respective levels at time zero. Data were derived from at least three independent experiments. The dotted line demarcates 50%. P-values from comparison of the decay rates are presented with asterisks. (***) P < 0.01 (two-tailed extra sum-of-squares F-test).

FIGURE 3.

Nudt3 is involved in reducing MCF-7 cell migration. (A) Immunofluorescence of filamentous (F)-actin (red) and DNA (blue) in proliferating Con^KD and Nudt3^KD MCF-7 cells. (B) Motility of Con^KD or Nudt3^KD MCF-7 cells complemented with the indicated proteins were analyzed by an in vitro scratch-wound healing assay. The cells were cultured to confluent cell monolayers, treated with mitomycin C to inhibit cell division, scratched and photographed at 0, 8, and 24 h after wounding. (C) Proliferation of Con^KD and Nudt3^KD MCF-7 cells was quantified by counting the number of cells at day 1, 2, 3, and 4 after initial seeding of 1 × 10⁵ cells per 35-mm dish. Data are representative of three independent experiments. (D,E) The percentage of gap closure for Nudt3^KD and Nudt4^KD cells were quantitated with ImageJ software. At least five different wounds were quantified for each time point. Data are representative of three independent experiments ±SD. P-values are denoted by asterisks. (***) P < 0.001 (Student's t-test).

FIGURE 4.

Elevated ITGB6 expression in Nudt3 knockdown cells is essential for increased MCF-7 cells motility. (A) A representative Western blot of integrin β6 (ITGB6) protein in cell extract from Con^KD and Nudt3^KD MCF-7 cells is shown. (B) Cell migration of Con^KD and Nudt3^KD MCF-7 cells in the presence of 10 μg/mL of either αvβ6-neutralizing antibody (10D5) or nonspecific antibody (IgG2a) in the medium lacking FBS was assessed by scratch-wound healing assays. At least five different wounds were quantified for each time point. Data are representative of three independent experiments ±SD. P-values are denoted by asterisks. (***) P < 0.001 (Student's t-test).

FIGURE 5.

Increased FN secretion in cells with down-regulated Nudt3 is essential for increased MCF-7 cell motility. (A,B) FN protein secreted into the medium was isolated by gelatin–sepharose chromatography from cells grown without FBS on tissue culture plastic. Levels of secreted FN in Con^KD and Nudt3^KD cells and the same cells with down-regulated FN (A) or complemented with GFP, Nudt3, or Nudt3^EE/QQ expression constructs (B) were determined by Western blot analysis. (C) Migration of cells depicted in A were determined by scratch-wound healing assays. At least five different wounds were quantified for each time point. Data are representative of three independent experiments ±SD. P-values are denoted by asterisks. (***) P < 0.001 (Student's t-test).