The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF 65 and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF 65. Our results indicate that hUAP56 first interacts with U2AF 65 in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome. Pre-mRNA splicing occurs in a ribonucleoprotein (RNP) complex called the spliceosome, which comprises a large number of proteins and several U small nuclear RNP particles (snRNPs) termed U1, U2, U4, U5, and U6. Assembly of the spliceosome on the pre-mRNA substrate proceeds in a stepwise manner and involves the recognition of intron-defining splice signals (for review, see Hertel and Graveley 2005). Spliceosome assembly is initiated by the formation of complex E, in which U1 snRNP is stably associated with the 5Ј splice site, SF1 is associated with the branchpoint, and U2 snRNP auxiliary factor (U2AF) subunits U2AF 65 and U2AF 35 are associated with the polypyrimidine (Py)-tract and 3Ј splice site, respectively. Subsequently, SF1 is replaced by U2 snRNP at the branchpoint, leading to the formation of complex A (the prespliceosome). Mature spliceosome assembly occurs upon entry of the U4/U6 и U5 tri-snRNP to form complex B, followed by structural rearrangements to form the catalytically active complex C, in which U2 and U6 snRNAs interact, and U6 replaces U1 at the 5Ј splice site.Many steps in spliceosome assembly require ATP hydrolysis and are mediated by a series of splicing factors that are members of the DExD/H-box protein family, the founding member of which is eIF-4A, a known RNA helicase (for review, see Staley and Guthrie 1998;Silverman et al. 2003;Rocak and Linder 2004). Several of these DExD/H-box splicing factors have been shown to possess an ATP-dependent RNA unwinding/helicase activity (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Wagner et al. 1998;Wang et al. 1998) and are thought to use ATP hydrolysis as a driving force to modulate specific RNA structural rearrangements during spliceosome assembly. DExD/H-box proteins typically have a series of conserved sequence motifs; structural, mutational, and biochemical analyses have suggested roles for these motifs in ATP-binding, ATP hydrolysis (ATPase), RNA-binding, and dsRNA unwinding/hel...

The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 39 splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 39 splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2-and U12-type intron splicing.Supplemental material is available at http://www.genesdev.org.Received July 26, 2010; revised version accepted September 10, 2010.Pre-mRNA splicing occurs in a dynamic ribonucleoprotein (RNP) complex termed the spliceosome, which is composed of numerous proteins and small nuclear RNP particles (snRNPs). For the major class of introns, called U2-type introns, splicing occurs in a spliceosome that contains four U snRNPs: U1, U2, U5, and U4/U6 snRNPs (Black 2003). A small subset of introns, called U12-type introns, is spliced through the conventional two-step pathway, but by a different spliceosome that contains U5, U11, and U12 snRNPs, and an alternative form of U4/U6 snRNP called U4atac/U6atac (Will and Luhrmann 2005). On U12-type introns, the 59 and 39 splice sites and branchpoint are highly conserved and differ from those of the conventional U2-type introns (Hall and Padgett 1994;Sharp and Burge 1997), and the characteristic polypyrimidine (Py) tract is typically absent (Burge et al. 1998).Spliceosome assembly of U2-type introns is initiated by binding of U2AF to the Py tract/39 splice site. U2AF is a heterodimer composed of a large (65-kDa) and a small (35-kDa) subunit (Zamore et al. 1992). The large subunit, U2AF65, binds specifically to the Py tract, whereas the small subunit, U2AF35, contacts the AG dinucleotide at the 39 splice site (Merendino et al. 1999;Wu et al. 1999;Zorio and Blumenthal 1999). This contact results, at least in part, from a sequence-specific RNA-binding activity of U2AF35 that recognizes the 39 splice site AG. For introns with weak Py tracts, the U2AF35-39 splice site interaction is critical for U2AF binding and splicing.Genome sequence analysis and expression studies have revealed the existence of several U2AF35-related proteins (U2AF35-RPs). Each U2AF35-RP contains a common core, consisting of a noncanonical RNA recognition motif called the U2AF homology motif (UHM) (Kielkopf et al. 2004) and two flanking zinc finger domains, but differs at the N and/or C termini. In mammals, there are at least three U2AF35-RPs: U2AF26 and two highly similar proteins, U2AF1-RS1 and U2AF1-RS2 (also called Urp) (Kitagawa et al. 1995;Tronchere et al. 1997). U2AF26 is nearly identical to U2AF35, but lacks the C-terminal arginine-serine-rich (RS) domain that is present in U2AF35 (Shepard et al. 2002). U2AF26 associates with U2AF65 and can functionally substitute for U2AF35 in both constitutive and enhancer-...

Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of theTC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either submaximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl isoforms, and CrkII are not required components of insulin signaling to GLUT4 transporters.Glucose homeostasis is a tightly controlled process in mammals, requiring rapid disposal of ingested glucose into muscle and adipose tissue during high caloric intake and enhanced production of glucose by liver and kidney during starvation (1). Insulin is secreted during feeding and acts to stimulate the former process and inhibit the latter, thus maintaining appropriately low blood glucose concentrations (2). Insulin action to enhance glucose transport into muscle and fat cells reflects its ability to cause translocation of intracellular GLUT4 glucose transporter proteins to the plasma membrane (3, 4). The recycling of GLUT4 from intracellular compartments to the plasma membrane occurs in the basal state as well but at a much slower rate, and insulin action markedly enhances the exocytic pathway while inhibiting endocytosis (5). Insulin signaling to GLUT4 is dependent on the activation of the PI 1 3-kinase signaling pathway whereby generation of D-3 polyphosphoinositides leads to the activation of downstream protein kinases such as Akt (2, 6 -11). This cascade involves tyrosine phosphorylation of insulin receptor substrates and the recruitment of the p85 regulatory subunit of PI 3-kinase to these phosphotyrosines through binding of its SH2 domains (12-16). Disruption of PI 3-kinase activation by insulin through the action of inhibitors or dominant inhibitory constructs blocks GLUT4 translocation (17)(18)(19).Despite the clear dependence of GLUT4 regulation by insulin on PI-3 kinase activity, several lines of evidence have indicated that an exclusive PI 3-kinase-dependent pathway might be insufficient for glucose uptake in adipocytes. Thus, a membrane-permeant analog of the PI 3-kinase product phosphatidylinositol 3,4,5-trisph...

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(ii) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.

U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF 65 stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF 65 . U2AF 65 overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF 65 inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF 65 effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF 65 even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF 65 possesses a splicing Inhibitory function that leads to alternative exon skipping.U2AF 65 | pre-mRNA splicing | splicing inhibition | exon exclusion | SMN P re-mRNA splicing is a process in which noncoding intron sequences are removed and exon sequences are then ligated together (1, 2). Pre-mRNA splicing is carried out by spliceosome, a large RNA-protein complex that contains five small nuclear ribonucleoproteins (U snRNPs) and more than 100 additional proteins (3). Pre-mRNA splicing occurs in the consensus sequences at the 5′ splice-site, 3′ splice-site, and branch point that are necessary for splicing. The sequence between 3′ AG dinucleotide and branch point is the polypyrimidine (Py) tract that directs spliceosome assembly on the 3′ splice-site. Alternative splicing provides an important regulatory mechanism in higher eukaryotes for multiple proteins produced from a single gene (4, 5).The U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) exists as a heterodimer with U2AF 35 (6). U2AF 65 contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/ serine-rich (RS) domain (7,8). Using U2AF 65 depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF 65 is an essential splicing factor (9). Whereas U2AF 65 binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF 35 plays a role in the 3′ splice-site (10, 11). As U2AF 65 prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF 65 and Py tract promotes prespliceosome assembly (12). U2AF 65 is also essential in vertebrate development (13,14). Its expression level is related to myotonic dystrophy, cystic fibrosis, and cancers (15, 16).Proximal spinal muscular atrophy (SMA) is an autosomal recessive genetic disease (17) and a leading cause of infant mortality. The motor neurons in the anterior horn of spinal cord are severely damaged in patients with type 1 SMA, usually leading to death before age 2 y as a result of a lack of respiratory support (18,19). In patients with SMA, the SMN1 gene is deleted or mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, is included (20). SMN2 genomic DNA...