The data are presented as a percentage of gap closure over time. Other Materials. of the inositol pyrophosphate (PP-InsP) signaling family, 5-diphosphoinositol pentakisphosphate (5-InsP7; Fig. 1and and and Knockout Cells. The knockout (KO) of KO cells. Thus, we have used these cells as a model for exploring if there is a role for 5-InsP7 in regulating mRNA levels. In order to monitor NUDT3 activity in intact cells, we assayed the levels of a cadre of its preferred substrates: mRNAs for integrin 6 (ITGB6), fibronectin (FN1), lipocalin-2 (LCN2), and S100 calcium-binding protein A8 (S100A8) (2, 4). These four transcripts were identified by RNA-sequencing analysis to be among those that were the most responsive (in terms of elevated levels) upon stable knockdown of NUDT3 in an MCF-7 breast cancer cell line (4). That phenotype was complemented by overexpression of WT NUDT3 but not by the decapping-deficient NUDT3EE/QQ mutant (4). The latter work has also contributed to the current consensus that mammalian cells contain multiple decapping enzymes that each control the stability and expression of distinct mRNA transcripts (2, 3). Using quantitative real-time PCR, we found elevated levels of mRNA transcripts for ITGB6, FN1, LCN2, and S100A8 in KO HEK293 cells compared with WT cells (Fig. 1 KO cells contain similar levels of the ITGAV mRNA transcript (Fig. 1KO cells of the levels of mRNAs that are NUDT3 substrates is not due to a decrease in expression of NUDT3 itself (Fig. 1KO HEK293 cells. However, by themselves, these data do not exclude the alternate possibility that, through some unknown NUDT3-independent mechanism, 5-InsP7 may affect P-body accumulation (see below) and indirectly stabilize those mRNAs which are normally decapped by NUDT3. This possibility is hard to exclude SN 2 using most cell models, since NUDT3 knockdown and/or overexpression would be expected to impact levels of both 5-InsP7 levels and those mRNA transcripts that are decapped by NUDT3. However, we have found an experimental system in which the 5-decapping and 5-InsP7 phosphatase activities of NUDT3 are uncoupled: the MCF-7 model in which NUDT3-mediated 5 decapping Rabbit Polyclonal to Synapsin (phospho-Ser9) was first established (see above). We used high-performance liquid chromatography analysis of [3H]inositol-labeled WT and NUDT3 knockdown MCF-7 cells to quantify 5-InsP7 levels, and found there was not a significant difference between the two cell lines (> 0.4): WT cells, 5-[3H]InsP7, 3.9 0.6 10?3 (relative to [3H]InsP6) and 1.2 0.2 SN 2 10?5 (relative to SN 2 [3H]inositol lipids), = 4; the corresponding data for NUDT3 knockdown cells are 4.8 1 10?3 and 1.4 0.2 10?5, respectively. It is also notable that the levels of 5-InsP7 in WT cells are almost 10-fold less than the usual value for mammalian cells (i.e., the corresponding value for HEK293 cells is 3.0 0.2 10?2; Fig. 1KO HCT116 cells, in which levels SN 2 of InsP7 are also elevated (9). We found that these KO cells also expressed higher levels of mRNA transcripts for ITGB6, FN1, LCN2, and S100A8, as compared with WT cells (KO did not affect levels of ITGAV mRNA (KO HCT116 cells were not associated with a general elevation in the levels of expression of the corresponding proteins, with the exception of FN1 (KO Cells. To pursue the idea that it is a higher 5-InsP7 concentration that promotes increased levels of NUDT3 mRNA substrates in KO cells, we used small interfering RNA (siRNA) to knock down IP6K-mediated 5-InsP7 synthesis (KO HEK293 cells (KO HCT116 cells. WT cells (blue) and two independent clones.
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