Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation.

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

After more than a decade, it is still unknown whether the plasmon-mediated growth of silver nanostructures can be extended to the synthesis of other noble metals, as the molecular mechanisms governing the growth process remain elusive. Herein, we demonstrate the plasmon-driven synthesis of gold nanoprisms and elucidate the details of the photochemical growth mechanism at the single-nanoparticle level. Our investigation reveals that the surfactant polyvinylpyrrolidone preferentially adsorbs along the nanoprism perimeter and serves as a photochemical relay to direct the anisotropic growth of gold nanoprisms. This discovery confers a unique function to polyvinylpyrrolidone that is fundamentally different from its widely accepted role as a crystal-face-blocking ligand. Additionally, we find that nanocrystal twinning exerts a profound influence on the kinetics of this photochemical process by controlling the transport of plasmon-generated hot electrons to polyvinylpyrrolidone. These insights establish a molecular-level description of the underlying mechanisms regulating the plasmon-driven synthesis of gold nanoprisms.

Abstract-Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. Nur77 is highly expressed in vascular endothelial cells (ECs) and plays a role in the regulation of cell proliferation and angiogenesis; its role in vascular inflammation, however, remains unknown. Treatment of human umbilical vein ECs (HUVECs) with tumor necrosis factor (TNF)-␣ substantially increased the transcription and protein expression of Nur77 in a dose and time-dependent manner, as determined by Northern blot and Western blot analysis. Adenovirus mediated overexpression of Nur77 markedly increased the intracellular levels of IB␣ by approximately 4-fold, whereas overexpression of dominant negative Nur77 (DN-Nur77), which lacks its transactivation domain, had no effect on IB␣ expression, suggesting that Nur77 is an important transcriptional factor in controlling IB␣ expression in ECs. Furthermore, overexpression of Nur77 significantly increased IB␣ promoter activity via directly binding to a Nur77 response element in the IB␣ promoter. Importantly, overexpression of Nur77, but not DN-Nur77, protected ECs against the TNF-␣-and interleukin-1␤-induced endothelial activation, as characterized by attenuation in the nuclear factor B activation, expression of adhesion molecules ICAM-1 and VCAM-1, and monocytic adherence to ECs.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.

Abstract-Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-␣ at the posttranscriptional level. To elucidate the molecular basis of TNF-␣-mediated eNOS mRNA instability, eNOS 3Ј untranslated region (3Ј-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-␣-stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3Ј-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-␣. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-␣ 1 (eEF1A1). In HUVECs, TNF-␣ rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase-eEF1A1 fusion protein specifically binds to a UC-rich sequence in the 3Ј-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3Ј-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3Ј-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-␣-induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3Ј-UTR binding protein that plays a critical role in mediating TNF-␣-induced decrease in eNOS mRNA stability. (Circ Res. 2008;103:591-597.)Key Words: TNF-␣ Ⅲ eNOS Ⅲ mRNA Ⅲ stability Ⅲ translation elongation factor 1-␣ E ndothelial nitric oxide synthase (eNOS) is a key enzyme involved in the regulation of vascular function and abnormality of eNOS activity and/or expression has been shown to cause several vascular diseases. 1 Although eNOS was initially considered to be a constitutive enzyme, it was shown later that eNOS expression was regulated by a variety of exogenous stimuli. For instance, in cultured endothelial cells, cytokines, lipopolysaccharide, and oxidized LDLs have been shown to downregulate eNOS expression. 2,3 In contrast, shear stress, estrogen, and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors have been demonstrated to upregulate eNOS expression. 4 -6 For many of these stimuli, modulation of eNOS mRNA stability plays an essential role in the regulation of eNOS expression. 7 However, the mechanism(s) responsible for the regulation of eNOS mRNA half-life remains to be determined.One of the most potent inhibitory stimuli for eNOS expression in vascular endothelial cells is tumor necrosis factor (TNF)-␣. TNF-␣-mediated inhibition of eNOS expression, via a mechanism of destabilization of eNOS mRNA, has been shown to ...

Severe acute respiratory syndrome–associatedcoronavirus (SARS-CoV) was isolated from a pig during a survey for possible routes of viral transmission after a SARS epidemic. Sequence and epidemiology analyses suggested that the pig was infected by a SARS-CoV of human origin.

Background:The widespread threat of severe acute respiratory syndrome (SARS) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. Consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from SARS-coronavirus structural proteins. Methods: We synthesized 41 peptides ranging in size from 16 to 25 amino acid residues of relatively high hydrophilicity. The immunoreactivities of the peptides with SARS patient sera were determined by ELISA. Results: Four epitopic sites, S599, M137, N66, and N371-404, located in the SARS-coronavirus S, M, and N proteins, respectively, were detected by screening synthesized peptides. Notably, N371 and N385, located at the COOH terminus of the N protein, inhibited binding of antibodies to SARS-coronavirus lysate and bound to antibodies in >94% of samples from SARS study patients. N385 had the highest affinity for forming peptide-antibody complexes with SARS serum.