Summary MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNP (microRNA-protein complex) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling and therefore, reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation and thereby, for the oocyte immature state. P97 interacts with 3′-UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5′ caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.

The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT] ex ) is elevated. It is well reported that stimulation of cells with high [5HT] ex induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT] ex , Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616 -624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT] ex "paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation.The serotonin transporter (SERT) 2 is a member of the Cl Ϫ -and Na ϩ -dependent monoamine transporter family, which also includes the dopamine transporter (DAT) and the norepinephrine transporter. SERT is a 630-amino acid plasma membrane-bound glycoprotein. Hydropathy analysis predicts that SERT contains 12 transmembrane domains and that both the N and C termini are exposed to the cytoplasm. The primary function of SERT in the central nervous system involves the regulation of serotonergic signaling via transport of serotonin (5-hydroxytryptamine (5HT)) molecules from the synaptic cleft into the pre-synaptic terminal for re-utilization. SERT is also expressed in non-neuronal cells, including platelets, placental, intestinal and adrenal cell lines, but the exact function of SERT in these cell lines is still under investigation (1-5).The C-and N-terminal regions of monoamine transporter proteins have just recently garnered increased attention for their importance in transport function and localization. Significant work has been accomplished in identifying the importance of the C-terminal region of DAT and norepinephrine transporter in transporter function, expression, and localization (6 -9).The proteins interacting with the N terminus of SERT are syntaxin 1A (10, 11) and secretory carrier membrane protein 2 (12). SERT also complexes with Hic-5 (13) and ␣-synuclein (14), but the functional significance of these interactions are not known. PICK1 (15), MacMARCKS (16), the actin cytoskeleton (49), neuronal nitric-oxide synthase, and Se...

Background: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. Results:We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo.Conclusions: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.