U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

The emerging role of alternative splicing in senescence and aging

Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age‐related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF‐1, SIRT1, and ING‐1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype.     

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.     

Proteolysis of splicing factors during rat and monkey cell fractionation.

We have investigated the ability of various rat and monkey cell lines to yield nuclear extracts that would allow splicing of a model adenovirus pre-mRNA substrate. Extracts from normal FR3T3, rat-1 and CV-1 fibroblasts were unable to assemble splicing complexes and displayed a dramatic reduction in the binding activity of the splicing factor 65 kD U2AF. These results correlated with reduced levels of 65 kD U2AF and the snRNP-associated B protein. When a battery of protease inhibitors was used during cell fractionation, increased levels of 65 kD U2AF and B proteins were detected. Most importantly, U2AF binding and complex formation were dramatically improved in FR3T3, rat-1 and CV-1 extracts. Interestingly, transformation of rat and monkey cells with the SV40 large T antigen yielded extracts active in complex formation. Similar extracts were generated following transformation of rat-1 cells with the Py middle T antigen but not with the v-fos oncogene. Only SV40-transformed FR3T3 extracts displayed splicing activity. Our results indicate that proteolysis is a major obstacle encountered during the preparation of active extracts from normal rat and monkey cells and suggest that cells transformed with T antigens manifest reduced proteolysis during fractionation.     

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature and Aging Effects on Leaf Membranes of a Cold Hardy Perennial, Fragaria virginiana

The lipid composition of leaves of wild strawberry (Fragaria virginiana Duchesne) was analyzed throughout an annual growth cycle in the field. Cellular hardiness to temperature stress was assessed concomitantly by a solute leakage technique. Leaves were shown to be very sensitive to an applied temperature of −5°C during the summer months but insensitive to a 35°C treatment. This general pattern was also seen in young overwintering leaves but was reversed after a period of low-temperature hardening of these same leaves. Associated with cold hardening of the overwintering leaves was a twofold increase in the phospholipid content of the leaf membranes with a proportionately smaller increase in free sterols. The large increase in phospholipids presumably is due primarily to the proliferation of a sterol-poor membrane fraction, probably the endoplasmic reticulum. These quantitative changes in membrane material may be important in increasing freezing tolerance in the overwintering leaf cells by enhancing the overall capacity of the cell for plasma membrane and tonoplast extension through vesicle fusion using components from this endomembrane pool. Analysis of electron micrographs of hardened leaf cells showed an increase in vesiculated smooth endoplasmic reticulum and tonoplast membrane over nonhardened leaf cells, the latter resulting in an enhanced tonoplast surface area to vacuolar volume ratio. During this same period, no changes in the fatty acid or free sterol composition were detectable, suggesting that regulation of membrane fluidity via these components is not required for cold acclimation in this species. During aging and senescence of both the overwintering and the summer leaves, the cellular membranes remained functionally intact but became progressively more vulnerable to temperature stress. Free sterol content increased during this time. This feature may be related to the inability of the older leaves to withstand environmental stress. Increasing sensitivity of the cellular membranes to stress may, in turn, be causally related to the actual onset of senescence in these leaves, thus explaining why only the older leaves senesce when the plant is challenged by periodic environmental stress.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.

The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing.     

Thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse

Summary The effects of the administration of thyroxine (T4) on the postnatal cytodifferentiation of granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of Lewiss-Webster mice were studied by light and electron microscopy. From birth, mice of both sexes were injected daily with T4 (sc 0.4 g/g BW) and were sacrificed 24 h after the last injection at 7, 9, 11, 14 and 21 days of age. Control mice received vehicle only. In control mice, granulated striated duct (SD) cells were first detected at 9 days and 7 days of age by light- and electron microscopy, respectively. Furthermore, a few scattered granulated SD cells were observed by light microscopy as early as day 7 in T4-treated mice of both sexes. At 21 days of age, in mice given T4, GCT cells were larger and more numerous and the Golgi apparatus, rough endoplasmic reticulum, and secretion granules were more abundant. In control mice, immunocytochemical staining for epidermal growth factor-(EGF) was first detectable at day 21 at the light- and electron-microscopic levels. However, positively stained cells were first observed in T4-treated mice by light- and electron-microscopic immunocytochemistry at 14 and 11 days of age, respectively. Moreover, in the 21-day-old T4-treated mice, the number of immunoreactive GCT cells, as well as the intensity of the staining per cell, was markedly increased as compared to controls. EGF immunostaining was restricted to GCT cells, and by immuno-electron-microscopy was only seen in apical secretory granules in granulated SD cells and GCT cells. There were no sex differences in the differentiation of the duct system under any conditions. It is concluded that T4 stimulates the biosynthesis of EGF by an acceleration of the differentiation of the GCT precursor cells to mature cells.Supported in part by grant no. MT-5730 from the Medical Research Council of CanadaHolder of a fellowship from the Medical Research Council of CanadaScholar of the Fonds de la Recherche en Santé du Québec