The Wiskott-Aldrich syndrome protein (WASp) couples actin cytoskeletal rearrangement to T cell activation, but the mechanisms involved are unknown. Here, we show that antigen-induced formation of T cell:APC conjugates and synapses is abrogated in WASp-deficient T cells and that CD2 engagement evokes interactions between the proline-rich region required for WASp translocation to the synapse and the PSTPIP1 adaptor SH3 domain and between the PSTPIp1 coiled-coil domain and both CD2 and another CD2-binding adaptor, CD2AP. The induced colocalization of these proteins at the synapse is disrupted by expression of coiled-coil domain-deleted PSTPIP1. These data, together with the impairment in CD2-induced actin polymerization observed in WASp-deficient cells, suggest that PSTPIP1 acts downstream of CD2/CD2AP to link CD2 engagement to the WASp-evoked actin polymerization required for synapse formation and T cell activation.

The hydrolysis of ATP by a group of RNA-dependent ATPases (DEAD/H proteins) is required for spliceosome assembly, but not for the subsequent transesterification reactions. Little is known about the function of these ATPases in relation to the RNA conformational changes that occur in formation of active structures, in which U2/U6 small nuclear RNA (snRNA) interactions are essential for splicing to take place. Using a synthetic lethal genetic screen, we have isolated four yeast splicing factors involved in U2/U6 snRNA interactions (D.X. et al., manuscript in preparation). The RNA-dependent ATPase activity associated with one such factor, the Slt22 protein, is stimulated preferentially by annealed U2/U6 snRNAs. Both mutant slt22-1 and U2 snRNA cause a reduction in stimulation. The slt22-1 mutation blocks splicing at or before the first step, resulting in the accumulation of an unusual complex which lacks U5 snRNA. Our results indicate that the U2/U6 snRNA interactions facilitated by Slt22 are also involved in the interaction of U5 snRNA with the spliceosome.

Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in bronchial epithelial cell lines. Treatment of these cells with IL-1β and TNF-α resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-κB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-κB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in Elf3 (homologous to human ESE-1) knockout mice, the expression of the inflammatory cytokine interleukin-6 (IL-6) is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

The interaction of nocodazole with calf brain tubulin was studied to determine the effect of such interaction on the structure of tubulin. The effect of nocodazole on the self-association of tubulin was monitored by turbidity measurements and velocity sedimentation. Sedimentation patterns indicate that nocodazole neither induces tubulin to undergo self-association to form higher orders of aggregate nor does it perturb the equilibrium of the reaction leading to the formation of 42S double-ring structures although nocodazole binds to both the tubulin dimers and the polymeric form. Nocodazole does, however, inhibit the in vitro reconstitution of microtubules, and the presence of microtubule-associated proteins does not amplify the inhibitory effect of the drug. The conformational changes in tubulin upon binding of nocodazole were monitored by differential spectroscopy, circular dichroism, fluorescence, and chemical modification of sulfhydryl residues. Results from these studies show that the sulfhydryl residues become more accessible to chemical modification. In contrast, the binding of nocodazole does not significantly alter the net environment of tryptophan chromophores. These residues are apparently not all located on the surface of the tubulin molecule and at least some are partially buried.

Calf brain tubulin was subjected to isoelectric focusing and tryptic peptide map analysis. Results from isoelectric focusing experiments showed a total number of 17 well-resolved protein peaks. The number of peaks and the mass distribution under each peak remained the same when the concentration of protein or ampholyte was altered. When the protein was subjected to two-dimensional isoelectric focusing, a diagonal pattern was observed, indicating that the multiple peaks observed are not a manifestation of tubulin- ampholyte interaction. Further investigation by isolating these individual subspecies and subjecting them to isoelectric focusing yielded single peaks corresponding to the original ones without generating the initial pattern of multiple peaks. Tryptic peptide maps showed that among the subspecies of the alpha subunit there are 26 spots that are common among them. There are, however, 7 +/- 1 spots that are unique in each subspecies. Similar observations were obtained for the subspecies of the beta subunit although there are only 2 +/- 1 unique spots in each subspecies. These results suggest that tubulin subunits probably consist of polypeptides with both constant and variable regions in their sequences. Identical results were obtained for canine and rabbit brain tubulin, indicating that tubulin polymorphism is common among brain tissues. Tubulin isolated by either the polymerization-depolymerization or the modified Weisenberg procedures yielded identical results. These results show that the same subspecies of tubulin are extracted by both isolation procedures.

Base-pairing between U2 and U6 snRNAs to form intermolecular helix II has been demonstrated previously as a requirement for pre-mRNA splicing in mammalian cells. In contrast, deletion and substitution mutation experiments in yeast have indicated that helix II is not essential; instead, other regions of U2 and U6 have been proposed to pair, forming a helix called Ib. To investigate the importance of U2/U6 helices in yeast, we have systematically mutagenized the regions proposed to form helices II and Ib. Allele-specific suppression of certain U6 mutations by complementary substitutions in U2 shows that helix II indeed form in yeast but that it is essential only in the presence of additional mutations that disrupt U2 stem I and the proposed helix Ib. Similarly, the proposed helix Ib is essential only when helix II is disrupted. These observations provide an explanation for apparently conflicting data in yeast and mammalian experimental systems, and identify synergistic or functionally redundant interactions between U2 and U6 snRNAs.

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5 end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3 end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.Precursor-mRNA (pre-mRNA) splicing takes place in the spliceosome through a two-step transesterification reaction. At least 40 splicing factors have been identified by genetic means in the yeast Saccharomyces cerevisiae. Most have been implicated in specific steps of the splicing pathway (31,41). During the process of spliceosome assembly, small nuclear RNAs (snRNAs) and the pre-mRNA substrate, in association with protein factors, undergo extensive conformational changes which establish RNA-RNA interactions that are important for both splicing reactions. Among these factors are the DExD or DExH proteins: RNA-dependent ATPases (possibly RNA helicases), which include Prp2p, Prp5p, Prp16p, Prp22p, Prp28p (26, 31), Prp43p (2), and Slt22p (also called Brr2p) (22,34,53). Their functions are essential for the formation and maintenance of RNA-RNA interactions in the spliceosome.With few exceptions (28,29,36), Watson-Crick base pairing is important for most RNA-RNA interactions in the formation of the spliceosome (26). In particular, intermolecular basepairing interactions that occur between U2 and U6 snRNAs, forming helices I and II (Fig. 1A), are likely...

We have examined the extent of brain tubulin heterogeneity in six vertebrate species commonly used in tubulin research (rat, calf, pig, chicken, human, and lamb) using isoelectric focusing, two-dimensional electrophoresis, and peptide mapping procedures that provide higher resolution than previously available. The extent of heterogeneity is extremely similar in all of these organisms, as judged by number, range of isoelectric points, and distribution of the isotubulins. A minimum of 6 a and 12 f3 tubulins was resolved from all sources. Even the pattern of spots on two-dimensional peptide maps is remarkably similar. These similarities suggest that the populations of tubulin in all of these brains should have similar overall physical properties. It is particularly interesting that chicken, which has only four or five 8-tubulin genes, contains approximately 12 P tubulins. Thus, post-translational modification must generate at least some of the tubulin heterogeneity. Mammalian species, which contain 15-20 tubulin DNA sequences, do not show any more tubulin protein heterogeneity than does chicken. This suggests that expression of only a small number of the mammalian genes may be required to generate the observed tubulin heterogeneity.Microtubules are protein filaments composed principally of a-and f-tubulin subunits. They are involved in a variety of cellular functions including mitosis, maintenance of cell shape, cell motility, intracellular transport, and secretion (1). It has long been thought that these diverse functions are not all performed by microtubules assembled from a homogeneous pool of identical tubulin subunits (2). Indeed, there is now compelling evidence from electrophoresis (3, 4), chromatography (5, 6), isoelectric focusing (7-15), protein sequencing (16,17), and DNA sequencing (18-20) that a-and f-tubulin subunits are actually populations of heterogeneous proteins.The nature, extent, and significance of tubulin heterogeneity are not fully understood. Substantial progress has been made in determining the nature of the heterogeneity: multiple, nonidentical tubulin genes have been identified in all the higher eukaryotes that have been examined and in several, possibly all, cases more than one gene is expressed (see ref.21 for a recent review). This implies that different gene products probably account for some of the heterogeneity. Posttranslational modification may account for the remaining heterogeneity: detyrosylation (22), aminoacylation (23), phosphorylation (24), glycosylation (25,26), and acetate-metabolite addition (27) have been reported, although it is not known whether these modifications occur in all organisms. The functional significance of the heterogeneity has been more difficult to determine. Many observations suggest a correlation between tubulin differences and particular functions: expression of tubulin genes is tissue dependent (28-33); the occurrence and distribution of tubulin proteins is tissue specific (34-39); specific tubulins are associated with distinct tubulin popu...