In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3 splice sites (3ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3ss A2 and 5 splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3ss A2 splicing is necessary for HIV-1 replication.In contrast to the alternative splicing of most cellular mRNA, splicing of retroviral mRNA results in the cytoplasmic accumulation of incompletely spliced and unspliced viral mRNA. Incompletely spliced viral mRNA is necessary for the expression of the Env, Vpu, Vpr, and Vif proteins, and the accumulation of unspliced viral mRNA is necessary for expression of the gag and pol gene products and also serves as the genomic viral mRNA encapsidated within progeny virions. Completely spliced viral mRNA is required for expression of the regulatory viral proteins Tat, Rev, and Nef. More than 40 unique incompletely and completely spliced viral mRNA species are generated through the alternative splicing of the HIV-1 primary transcript within an HIV-1-infected cell (23,26).HIV-1 3Ј splice sites (3Јss) are used with differing efficiencies in part because viral polypyrimidine tracts (PPT) are interspersed with purines (27, 30), leading to decreased affinity for the essential cellular splicing factor U2AF65 (for a review of cellular splicing see reference 12). In addition, the efficiency of HIV-1 splicing is also regulated by both positive and negative cis elements within the viral genome that act to promote or repress splicing. To date, four exonic splicing silencers (ESS) and one intronic splicing silencer (ISS) have been identified within the viral genome (Fig. 1). Utilization of HIV-1 3Јss A2 by the spliceosome is negatively regulated by ESSV, 3Јss A3 by ESSp and ESS2, and 3Јss A7 by the ISS and ESS3 (2,3,5,16,29,31).We have previously characterized ESSV as a 24-nucleotide

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of G␣ and G␤␥ subunits. Here we evaluated the role of G␤␥ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking G␤␥ signaling with G␣ t or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of G␣ t in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking G␤␥ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that G␤␥ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of G␤␥ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of G␤␥ signaling in promoting breast tumor growth and metastasis and suggest that targeting G␤␥ may represent a novel therapeutic approach for breast cancer.Breast cancer is the most frequently diagnosed cancer in women (1). Despite recent advances in its diagnosis and treatment with adjuvant targeted therapies, breast cancer remains the second most common cause of cancer death among women in the United States (2). As many as 90% of cancer deaths are caused by metastatic spread from primary tumors because the majority of currently marketed anticancer drugs have little effect on tumors at this stage. In light of this situation, efforts to better understand the mechanisms underlying tumor metastasis and to identify novel treatment strategies are warranted.

We investigate whether unpleasant environmental conditions affect stock market participants' responses to information events. We draw from psychology research to develop a new prediction that weather-induced negative moods reduce market participants' activity levels. Exploiting geographic variation in equity analysts' locations, we find compelling evidence that analysts experiencing unpleasant weather are slower or less likely to respond to an earnings announcement relative to analysts responding to the same announcement but experiencing pleasant weather. Price association tests find evidence consistent with reduced activity due to weather-induced moods delaying equilibrium price adjustments following earnings announcements. We also use our analyst-based research design to re-examine an existing prediction that unpleasant weather induces investor pessimism, and find evidence of both analyst pessimism and reduced activity in the presence of unpleasant weather. Together, our study provides new evidence that both extends and reaffirms findings of a relation between unpleasant weather and market activities, and contributes to the broader psychology and economics literature on the impact of weather-induced mood on labor productivity. JEL codes: G14; M41

Metastasis involves the invasion of cancer cells across both the extracellular matrix and cellular barriers, and an evolving theme is that epithelial-to-mesenchymal transition (EMT) may mediate invasive cellular behavior. Previously, we isolated and analyzed a subpopulation of PC-3 prostate cancer cells, TEM4-18, and found that these cells both invaded an endothelial barrier more efficiently and exhibited enhanced metastatic colonization in vivo. Transendothelial migration of these cells depended on expression of ZEB1, a known regulator of EMT. Surprisingly, these cells were much less invasive than parental PC-3 cells in assays that involve matrix barriers. Here, we report that TEM4-18 cells express significantly reduced levels of two subunits of laminin-332 (␤3 and ␥2) and that exogenous laminin-332, or co-culture with laminin-332-expressing cells, rescues the in vitro invasion phenotype in these cells. Stable knockdown of ZEB1 in prostate cancer cells up-regulated LAMC2 and ITGB4 mRNA and protein and resulted in a concomitant increase in Transwell migration. Using chromatin immunoprecipitation (ChIP), we show that ZEB1 directly interacts with the promoters of LAMC2 and ITGB4. These results provide a novel molecular basis for reduced laminin-332 observed in clinical prostate cancer specimens and demonstrate a context-dependent role for EMT in invasive cellular behavior.Metastasis involves the invasion of cancer cells across natural barriers such as basement membranes, interstitial matrix, and the endothelium. Considerable effort over the past decades has defined a number of mechanisms that contribute to the invasive behavior of metastatic cancer cells such as elevated secretion of matrix-degrading enzymes, altered expression of matrix components or their receptors, and increased motility in response to tumor microenvironmental cues. Epithelial-to-mesenchymal transition (EMT) 3 is a mechanism involved in multiple aspects of mammalian development, such as gastrulation and formation of the neural crest, whereby cells lose epithelial identity and gain the ability to move to distant sites in the organism and thus is an attractive paradigm for understanding metastasis (1). Although abundant experimental and some clinical evidence for EMT in cancer exists, the extent to which this mechanism contributes to metastasis remains controversial (2). Although EMT has been most thoroughly investigated in relation to its role in invasion of basement membranes and interstitial matrix, less is known about how it might be involved in later steps of metastasis such as extravasation and colonization (survival and proliferation at distant sites).We recently isolated a subpopulation of the PC-3 prostate cancer cell line, TEM4-18, that was proficient in transendothelial migration and displayed hallmarks of EMT (3). We found that TEM4-18 cells differentially express a dual zinc finger homeodomain transcription factor ZEB1 (also known as ZFHX1a, ␦EF1, or TCF-8), which represses a variety of epithelial genes in TEM4-18 cells and has bee...