Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the spliceosome, a macromolecular complex composed of small nuclear ribonucleoproteins (snRNPs) and many additional proteins. Spliceosome assembly and splicing catalysis occur in an ordered multistep process, which includes multiple conformational rearrangements (35). Spliceosomal snRNPs are assembled from snRNAs and protein components, the latter of which fall into two classes: snRNP-specific and common proteins. The common or canonical core proteins are also termed Sm proteins, specifically SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (10; reviewed in reference 9), which all share an evolutionarily conserved bipartite sequence motif (Sm1 and Sm2) required for Sm protein interactions and the formation of the heteroheptameric Sm core complex around the Sm sites of the snRNAs (3, 7, 29). Prior to this, the Sm proteins form three heteromeric subcomplexes: SmD3/SmB, SmD1/SmD2, and SmE/SmF/SmG (23; reviewed in reference 34). Individual Sm proteins or Sm subcomplexes cannot stably interact with the snRNA. Instead, a stable subcore forms by an association of the subcomplexes SmD1/SmD2 and SmE/SmF/SmG with the Sm site on the snRNA; the subsequent integration of the SmD3/SmB heterodimer completes Sm core assembly.In addition to the canonical Sm proteins, other proteins carrying the Sm motif have been identified for many eukaryotes. Those proteins, termed LSm (like Sm) proteins, exist in distinct heptameric complexes that differ in function and localization. For example, a complex composed of LSm1 to LSm7 (LSm1-7) accumulates in cytoplasmic foci and participates in mRNA turnover (4, 8, 31). Another complex, LSm2-8, binds to the 3′ oligo(U) tract of the U6 snRNA in the nucleus (1, 15, 24). Finally, in the U7 snRNP, which is involved in histone mRNA 3′-end processing, the Sm proteins SmD1 and SmD2 are replaced by U7-specific LSm10 and LSm11 proteins, respectively (20, 21; reviewed in reference 28).This knowledge is based primarily on the mammalian system, where spliceosomal snRNPs are biochemically well characterized (34). In contrast, for trypanosomes, comparatively little is known about the components of the splicing machinery and their assembly and biogenesis. In trypanosomes, the expression of all protein-encoding genes, which are arranged in long polycistronic units, requires trans splicing. Only a small number of genes are additionally processed by cis splicing (reviewed in reference 11). During trans splicing, a short noncoding miniexon, derived from the spliced leader (SL) RNA, is added to each protein-encoding exon. Regarding the trypanosomal splicing machinery, the U2, U4/U6, and U5 snRNPs are considered to be general splicing factors, whereas the U1 and SL snRNPs represent cis- and trans-splicing-specific components, respectively. In addition to the snRNAs, many protein splicing factors in trypanosomes have been identified based on sequence homology (for example, see references 14 and 19).Recent studies revealed variations in the Sm core compositions of spliceosomal snRNPs from Trypanosoma brucei. Specifically, in the U2 snRNP, two of the canonical Sm proteins, SmD3 and SmB, are replaced by two novel, U2 snRNP-specific proteins, Sm16.5K and Sm15K (33). In this case, an unusual purine nucleotide, interrupting the central uridine stretch of the U2 snRNA Sm site, discriminates between the U2-specific and the canonical Sm cores. A second case of Sm core variation was reported for the U4 snRNP, in which a single protein, SmD3, was suggested to be replaced by the U4-specific LSm protein initially called LSm2, and later called SSm4, based on a U4-specific destabilization after SSm4 knockdown (30). A U4-specific Sm core variation was also previously suggested and discussed by Wang et al. (33), based on the inefficient pulldown of U4 snRNA through tagged SmD3 protein. However, neither of these two studies conclusively demonstrated by biochemical criteria that the specific Sm protein resides in the U4 Sm core; a copurification of other snRNPs could not be unequivocally ruled out.By using a combination of RNase H cleavage, tandem affinity purification, and mass spectrometry, we provide here direct biochemical evidence that in the variant Sm core of the U4 snRNP, only SmD3 is replaced by the U4-specific SSm4. SSm4 is nuclear localized, and the silencing of SSm4 leads to a characteristic phenotype: dramatic growth inhibition, general trans- and cis-splicing defects, a loss of the integrity of the U4 snRNA, as well as a destabilization of the U4/U6 di-snRNP. Furthermore, in vitro reconstitution assays revealed that under stringent conditions, SSm4 is sufficient to specify U4-specific Sm core assembly. In sum, our data establish SSm4 as a specific component of the U4 Sm core and demonstrate its importance in U4/U6 di-snRNP biogenesis, splicing function, and cell viability.     

Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.     

Examining the sources of variability in cell culture media used for biopharmaceutical production

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

Response of the Cholesterol Metabolism to a Negative Energy Balance in Dairy Cows Depends on the Lactational Stage

The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.     

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific puffs in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5×10^–6 mol/l 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca²⁺-dependent increase of nuclear volume by 23.0±2.3%. Amiloride, a blocker of plasma membrane Na⁺/H⁺ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9±3.9%. Measurments of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23±0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amilorde-sensitive plasma membrane Na⁺/H⁺ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.     

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.