Search

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-β signals that block cell cycle progression in G1are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-β signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467 – sites on Smads that get phosphorylated by the TGF-β receptor and positively regulate TGF-β signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-β. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255 – sites that are phosphorylated by MAP kinase and negatively regulate TGF-β signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-β. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-β signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-β signals that promote G1arrest.