Heterogeneous Nuclear Ribonucleoprotein K Represses the Production of Pro-apoptotic Bcl-xS Splice Isoform

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x_L and the pro-apoptotic Bcl-x_S isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5′ splice site of Bcl-x_S. The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein. Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x_S, an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x_S splice isoform.Alternative splicing is a major mechanism used to augment the number of proteins encoded by the genome. It is estimated that as many as 97% of multiple exon pre-mRNAs undergo alternative splicing (1, 2). Disruption of alternative splicing by mutating important regulatory sequences or by altering the expression or activity of proteins controlling splice site selection has been linked with different diseases, including cancer (3–7). Apoptosis is an important and complex cellular program involved in development and differentiation in higher organisms (8, 9). However, its aberrant control often contributes to cancer development and the resistance of cancer cells to drug therapy (10–13).Genes implicated in the apoptotic pathway are alternatively spliced often to produce protein isoforms with distinct or even antagonistic activities (14, 15). A good example is the apoptotic regulator Bcl-x, which is alternatively spliced to produce two major isoforms, the anti-apoptotic Bcl-x_L protein and the shorter pro-apoptotic Bcl-x_S isoform (16). This alternative splicing decision involves a competition between two 5′ splice sites; the use of the downstream site creates Bcl-x_L, and the use of the upstream one produces Bcl-x_S (Fig. 1A). Bcl-x_L is always the predominant form in cancer cells, and overexpressing it can confer resistance to chemotherapeutic agents (17–22). On the other hand, overexpression of the pro-apoptotic Bcl-x_S isoform enhances sensitivity to the topoisomerase inhibitor etoposide and to taxol in a breast cancer cell line, while triggering apoptosis in melanoma cell lines (23, 24). Using antisense technologies to improve the production of the Bcl-x_S splice variant can also induce apoptosis in cancer cells (25–27).Open in a separate windowFIGURE 1.A, alternative splicing of Bcl-x produces two major isoforms, Bcl-x_L and Bcl-x_S. B, regulation of Bcl-x alternative splicing. The enhancer elements are shown as white boxes, and the repressors are black. The pointed and flat arrows indicate positive and negative regulation, respectively. Protein kinase C inhibition relieves repression caused by the SB1 element on the Bcl-x_S splice site (36). The repressor elements CRCE1, recognized by SAP155, and CRCE2 mediate the production of Bcl-x_S by ceramide as when induced by gemcitabine in A549 cells (38, 39). hnRNP F/H binds to the B2G element to enhance the production of the Bcl-x_S isoform (41). RBM25, through an element located upstream of the Bcl-x_S splice site, can also augment its use (44). A large intronic region (IRE) mediates the Bcl-x_L increase caused by interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (35). Finally, the B3 region also enhances Bcl-x_L formation through the binding of SRp30c to AM2 and ML2 and the U1 snRNP to two cryptic 5′ splice sites (42).Alternative splicing is regulated by different proteins bound to sequence elements near splice sites. A variety of mechanisms is used to achieve regulation. Some splicing factors act by recruiting or inhibiting the binding of different components of the spliceosome. Others may change the conformation of the pre-mRNA to mask a splice site or to bring a pair of splice sites into closer proximity (28, 29).Although individual factors can have a strong and specific effect on splicing decisions, alternative splicing often relies on a combination of factors to determine the appropriate levels of isoforms. The implication of multiple proteins likely provides additional levels of regulation that helps attuned splicing control to a variety of stresses, environmental cues, and growth conditions. In several cases, the interaction of regulatory factors can be antagonistic. For example, in the Drosophila male-specific-lethal-2 (msl-2) pre-mRNA, recruitment of SXL to a uridine-rich region interferes with the binding of TIA-1 that is necessary for efficient U1 snRNP² recruitment at the 5′ splice site (30). On the same pre-mRNA, SXL also diminishes U2AF recognition of the polypyrimidine tract at the 3′ splice site. TIA proteins bound to a U-rich element on the avian myosin phosphatase targeting subunit-1 (MYPT1) pre-mRNA repress the binding of PTB (31). PTB can also reduce the recruitment of ETR-3 to intronic elements near exon 5 of cardiac troponin T (32). In neurons, the binding of PTB to the introns surrounding the N1 exon of c-src is antagonized by nPTB protein, promoting exon inclusion. On the hnRNP A1 pre-mRNA, PTB diminishes the binding of SRp30c to the intronic CE9 element, reducing the inhibition by this protein on the use of the downstream 3′ splice site (33). SC35 and hnRNP A1 have partially overlapping binding sites on the human immunodeficiency virus 1 (HIV-1) tat exon 2. Preferential binding of SC35 enhances the inclusion of the exon, whereas hnRNP A1, by reducing SC35 binding, increases exclusion (34). Thus, the competition provided by an overlapping or a closely abutting pair of enhancer/ silencer represents a simple and frequent mechanism of splicing control.The regulation of Bcl-x alternative splicing has received some attention in recent years leading to the discovery of several cis-acting elements and a few trans-acting control factors (Fig. 1B). Intronic regions downstream from the Bcl-x_L 5′ splice site have been implicated as mediating signals from cytokines such as interleukin-6 and granulocyte-macrophage colony-stimulating factor (35). In addition, we have reported that an element located 187 nt upstream of the Bcl-x_S splice site mediates a protein kinase C-dependent signal that represses splicing to the Bcl-x_S donor site (36). On the other hand, ceramide enhances the use of the Bcl-x_S 5′ splice site by lifting the repression mediated by two other elements (37, 38). The activity of one of these apparently involves SAP155 (39). The RNA-binding protein Sam68, under the control of the tyrosine kinase Fyn, can also increase the production of Bcl-x_S in cooperation with hnRNP A1 (40), and this effect is inhibited by overexpression of ASF/SF2. The Bcl-x sequences bound by the above factors remain to be identified. We also uncovered enhancer elements for Bcl-x_S and Bcl-x_L. hnRNP F and H bind downstream of the Bcl-x_S 5′ splice site to stimulate splicing to that site (41). Enhancement of Bcl-x_L is conferred by SRp30c, which binds upstream of the 5′ splice site to antagonize the repressor activity of pseudo 5′ splice sites (42). Recently, the SR protein SC35 was shown to increase the production of Bcl-x_S (43). Finally, the binding of RBM25 to a sequence element upstream of the Bcl-x_S 5′ splice site stimulated its use, possibly by recruiting U1 snRNP through its interaction with the U1-associated protein hLuc7A (44). Thus, the region located between the two competing 5′ splice sites of Bcl-x is densely populated by splicing control elements.In this study, we have pursued our characterization of Bcl-x splicing control by examining the contribution of sequences directly upstream of the Bcl-x_S donor site. Our mutational approach identified a region containing flanking enhancer and silencer activities. The activity of the repressor portion is mediated by hnRNP K, which makes this protein an anti-apoptotic regulator.     

Isolation and characterization of 16 polymorphic microsatellite loci in the endemic Hawaiian damselfly Megalagrion xanthomelas (Odonata: Coenagrionidae)

Microsatellite loci have been isolated from two species of endemic Hawaiian damselflies, Megalagrion xanthomelas and M. eudytum, that are of conservation concern. Sixteen polymorphic loci were characterized in 32 M. xanthomelas from one population on Molokai and one on Hawaii Island. The total number of alleles per locus ranged from two to 16 and observed population heterozygosity ranged from 0.0 to 0.963. Eleven of these loci amplified successfully in M. eudytum as well. These loci will be used to further conservation efforts and infer genetic consequences of Pleistocene glaciations.     

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

Background

The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals.

Methodology/Principal Findings

We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst ∼8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs.

Conclusions/Significance

In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress.     

Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that Gα12 inhibits interaction of MDCK cells with collagen-I, the major ligand for α2β1 integrin. Activating Gα12 (QL point mutation or stimulating endogenous Gα12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with Gα12-regulated attachment to collagen-I, Gα12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in Gα12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. Gα12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of α2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated Gα12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, Gα12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for Gα12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.     

The influence of historical landscape change on genetic variation and population structure of a terrestrial salamander (Plethodon cinereus)

Forest loss and fragmentation is expected to shape the genetic structure of amphibian populations and reduce genetic variation. Another factor widely understood to have impacted these same parameters in North America is the range expansion that occurred following glacial retreat at the end of the Pleistocene. The Eastern Red-Backed Salamander (Plethodon cinereus) has been subjected to both processes. In this context, we investigated the historical events that are likely to have shaped genetic variation in this species using a panel of six microsatellite markers screened on individuals sampled across ten localities in northeastern Indiana, USA. We found low genetic diversity across forest patches and minimal differentiation. We expected population structure associated with forest fragmentation to result from genetic drift in isolation but instead found that a balance between gene flow and drift was ~50 times more likely. Ratios of allele number and range (M), and coalescent modeling of population demography suggested the occurrence of marked historic decline in effective population size across the region. Taken together, the data point to a loss of genetic variation which preceded deforestation over the past 200 years. This result indicates an important role for ancient demographic processes in shaping current genetic variation that may make it difficult to detect the effect of recent habitat fragmentation.     

The open state gating mechanism of gramicidin a requires relative opposed monomer rotation and simultaneous lateral displacement

The gating mechanism of the open state of the gramicidin A (gA) channel is studied by using a new Monte Carlo Normal Mode Following (MC-NMF) technique, one applicable even without a target structure. The results demonstrate that the lowest-frequency normal mode (NM) at approximately 6.5 cm(-1) is the crucial mode that initiates dissociation. Perturbing the gA dimer in either direction along this NM leads to opposed, nearly rigid-body rotations of the gA monomers around the central pore axis. Tracking this NM by using the eigenvector-following technique reveals the channel's gating mechanism: dissociation via relative opposed monomer rotation and simultaneous lateral displacement. System evolution along the lowest-frequency eigenvector shows that the large-amplitude motions required for gating (dissociation) are not simple relative rigid-body motions of the monomers. Gating involves coupling intermonomer hydrogen bond breaking, backbone realignment, and relative monomer tilt with complex side chain reorganization at the intermonomer junction.     

Depletion of a single nucleoporin, Nup107, induces apoptosis in eukaryotic cells

Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about on average 2,000 nuclear pore complexes (NPCs) in the nuclear envelope of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the NPC are known as nucleoporins. In mammalian cells, Nup107 is the homolog of yeast Nup84p nucleoporin. Nup107 contains a leucine zipper motif in its carboxyl-terminal region and numerous kinase consensus sites, but does not contain FG repeats. Previously it was reported that NUP88 and NUP107 are over expressed in many types of cancers including colon, breast, prostrate, etc. In this study, we were interested in investigating the role of NUP107 in grade 4 Astrocytoma, i.e., Glioblastoma multiforme cultured cell line. We transfected human Astrocytoma cells with Nup107-specific siRNA duplexes. The level of mRNA for Nup107 was monitored by RT-PCR, 24, 48, and 72 h after the initial transfection. Nup107 mRNA was significantly diminished by 24 h after transfection and we took that as our incubation time. Next we studied the effect of this inhibition on cell viability. We did a Trypan Blue cell viability assay and it showed increased cell death in NUP107 transfected cells than untreated control. We further tried to analyze the nature of the cell death whether apoptotic or necrotic by doing apoptosis assays like ssDNA ELISA assay, Caspase-3, and Caspase-8 assays. All the assays showed that siRNA transfected cells are undergoing increased apoptosis than control.     

Comparison of methods for measuring oxygen consumption in tumor cells in vitro

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.     

Triterpenoidal alkaloids from Buxus hyrcana and their enzyme inhibitory,anti-fungal and anti-leishmanial activities

From the aerial parts of Buxus hyrcana, three triterpenoidal alkaloids, 17-oxo-3-benzoylbuxadine (1), buxhyrcamine (2), and 31-demethylcyclobuxoviridine (3), along with 16 known compounds, cyclobuxoviridine (4), N_b-dimethylcyclobuxoviricine (5), E-buxenone (6), Z-buxenone (7), moenjodaramine (8), homomoenjodarmine (9), buxamine A (10), buxamine B (11), 31-hydroxybuxamine B (12), N₂₀-formylbuxaminol E (13), papillozine C (14), buxmicrophylline F (15), buxrugulosamine (16), cyclobuxophylline O (17), spirofornabuxine (18) and arbora-1,9(11)-dien-3-one (19) were isolated. Their structures were elucidated by using NMR spectroscopic methods. All of the compounds exhibited moderate to weak acetylcholinesterase, butyrylcholinesterase and glutathione S-transferase inhibitory activities. Compounds 1–19 also exhibited modest anti-fungal activities against Candida albicans. Compounds 1, 2, 8, 9 and 18 also exhibited weak anti-leishmanial activity.     

Oxo-bridged isomers of aza-trishomocubane sigma (σ) receptor ligands: Synthesis,in vitro binding,and molecular modeling

Isomeric oxo-bridged analogs of aza-trishomocubane sigma (σ) receptor ligands were synthesized and shown to display a reduced affinity for the σ receptor. In the case of phenethyl derivative 4, there was a concomitant introduction of high-affinity for the α_2C adrenergic receptor, and moderate affinity for the dopamine transporter. Molecular modeling was undertaken to rationalize these results.