HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are ''chaperoned'' by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a ''Y'' shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the ''Y'' conformation.HIV-1 nucleocapsid protein ͉ nucleic acid aggregation ͉ transactivation response element DNA/RNA annealing ͉ wild-type (WT)

Significance Misfolded proteins are a hallmark of diverse human cardiac disorders including desmin-related cardiomyopathy and systemic amyloidosis. Defects in translational fidelity can cause neurodegeneration, however the consequences of mistranslation in other tissues, including the heart, are unknown. The fidelity of protein synthesis is largely ensured by aminoacyl-tRNA synthetases, and many tRNA synthetases contain editing domains that hydrolyze mischarged tRNAs, preventing incorporation of incorrect amino acids into proteins. Here, we show that diminished editing efficacy of the alanyl-tRNA synthetase causes misfolded protein aggregation and cell death in the mammalian heart. These results illuminate the importance of translational fidelity in cardiac homeostasis and suggest that genetic factors that disrupt the accuracy of translation may contribute to proteinopathies of the heart and other tissues.

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.

SignificanceAminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs in the first step of protein synthesis in the cytoplasm. However, in higher eukaryotes, they acquired additional functions beyond translation. In the present study, we show that an activated form of tyrosyl-tRNA synthetase (YRSACT) functions to enhance megakaryopoiesis and platelet production in vitro and in vivo. These findings were confirmed with human megakaryocytes differentiated from peripheral blood CD34+ hematopoietic stem cells and with human induced pluripotent stem (iPS) cells. The activity of YRSACT is independent of thrombopoietin (TPO), as evidenced by expansion of the megakaryocytes from iPS cell-derived hematopoietic stem cells from a patient deficient in TPO signaling. These findings demonstrate a previously unrecognized function of an aaRS which may have implications for therapeutic interventions.