Decapping is a critical step in mRNA decay. In the 59-to-39 mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 59 to 39 exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 39-ends of mRNAs in vivo from trimming, presumably by binding to the 39-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 39-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNAbinding characteristics of this complex form a critical determinant of its in vivo interactions and functions.

Oxygen therapy often rescues and reduces the mortality resulting from acute respiratory distress syndrome, chronic obstructive pulmonary diseases, exposure to toxic fumes, and drowning (1). However, prolonged exposure to supra-physiological concentrations of oxygen, referred to as hyperoxia, causes extensive damage to the alveolar-capillary barrier resulting in increased permeability and decreased lung function (2). Although the molecular mechanisms of hyperoxia-induced lung injury and cell death are complex, recent studies suggest that the generation of excessive reactive oxygen species (ROS), 1 loss of antioxidant defense pathways, cytokine-mediated inflammation, and modulation of signal transduction may regulate pulmonary edema and apoptosis/necrosis of endothelial and epithelial cells (3). The vascular endothelium has long been recognized to generate superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( ⅐ OH), and nitric oxide (NO) via enzymatic and nonenzymatic reactions. In endothelial cells (ECs), in addition to the mitochondrial electron transport, other potential enzymatic pathways of ROS production include cyclooxygenase/lipoxygenase, cytochrome P450, xanthine oxidase, NADPH oxidase, NO synthase, and peroxidase. In the lung, the vascular NADPH oxidase seems to play an important role in excessive production of O 2 . in atherosclerosis, ischemic lung, pulmonary hypertension, and ventilator-associated lung injury (4 -9). NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to O 2 . by using NADPH or NADH as an electron donor (9). Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47 phox , p67 phox , and p40 phox ; a regulatory small molecular weight G-protein of either Rac1 or Rac2 and a membraneassociated cytochrome b 558 reductase made up of p22 phox and gp91 phox . We and others (10, 11) have shown that most of the subcomponents of phagocytic NADPH oxidase are expressed in vascular ECs. ECs exhibit a low output in of O 2. production under basal conditions, and stimulation by TNF-␣, pulsatile stretch, hypoxia reoxygenation, and phorbol ester enhanced the

The decapping of eukaryotic mRNA s is a key step in their degradation. The heteroheptameric L sm1p-7p complex is a general activator of decapping and also functions in protecting the 3Ј ends of deadenylated mRNA s from a 3Ј-trimming reaction. L sm1p is the unique member of the L sm1p-7p complex, distinguishing that complex from the functionally different L sm2p-8p complex. To understand the function of L sm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNAbinding and inter-subunit interaction residues of L sm1p led to impairment of mRNA decay, suggesting that the integrity of the L sm1p-7p complex and the ability of the L sm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the L sm1p-7p complex to the P-bodies; (iii) mRNA 3Ј-end protection could be indicative of the binding of the L sm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3Ј end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of L sm1p is also important for mRNA decay function.

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.