The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.
The detection of specific RNAs in a complex mixture of RNAs is typically accomplished by Northern blot (1). Here we report an alternative method for rapid and quantitative detection of RNA species, in which a radioactively-labeled oligonucleotide complementary to the RNA of interest is hybridized with the RNA in solution, and the resulting hybrid is resolved on a nondenaturing gel and detected by autoradiography. Because transfer to a membrane is not required and hybridization occurs in solution, our method is quicker and more quantitative than a Northern blot. Our application of this solution hybridization method to the small nuclear RNAs of the yeast Saccharomzyces cerevisiae demonstrates its specificity, sensitivity and ability to resolve similar RNA species. Figure 1A shows that 32P-labeled S. cerevisiae U6 snRNA (2) is quantitatively bound by an oligonucleotide (U6D) complementary to its 3' end after slowly cooling from 70°C. The protocol had no effect on the mobility of Xenopus laevis U1 snRNA, which was included in the hybridization mixture as a nonspecific RNA control. When 32P-labeled U6D oligo was annealed to unlabeled yeast total cellular RNA and applied to a nondenaturing gel, a single major hybrid species was detected (Figure iB). A band of identical mobility was seen when in vitro synthesized U6 snRNA was annealed to oligo U6D (data not shown), indicating that the U6D oligo is specific for U6 snRNA.The melting temperature (Tm) of the oligo U6D/U6 snRNA hybrid in hybridization buffer is 45°C. Incubation of the hybridization mixture at either 42°C or 37°C for 15 minutes instead of slowly cooling from 70°C gave efficient and specific hybrid formation, and was used in subsequent experiments. We have also used the solution hybridization method to detect U4 and U5 snRNAs ( Figure IC). Two U4 oligos shift the mobility of U4 snRNA (160 nt.) differently and bind with different efficiencies (Figure IC, lanes 1 and 2). The two bands seen in lane 3 presumably correspond to the long (L; 214 nt.) and short (S; 179 nt.) forms of US snRNA (3).Formation of hybrids at 370 to 42°C permits detection of the U4/U6 snRNA complex, which has a Tm of 53°C (4). In Figure ID, deproteinized yeast splicing extract was incubated with oligo U6D at 37°C for 15 minutes and applied to a nondenaturing gel. Both free U6 snRNA and U4/U6 snRNA complex were detected. As expected, the U4/U6 snRNA complex could also be detected with an oligo complementary to U4 RNA (data not shown).Our solution hybridization assay is sensitive and responds linearly to increasing amounts of RNA. One fmol of U6 snRNA (synthesized in vitro with T7 RNA polymerase) was easily detected (Figure 2A) and a plot of radioactivity in the hybrid band with 1 to 8 fmol of input U6 snRNA gave a straight line (correlation coefficient 0.994; Figure 2B). Therefore, quantitation of nonabundant RNAs can be accomplished with this method. From preliminary experiments we estimate that the average S. cerevisiae cell contains one to two thousand molecules of U6 snRNA.To test if sub...
The G protein ␥13 subunit (G␥13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for G␥13 by yeast two-hybrid and pulldown assays. In two-hybrid assays, G␥13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. G␥13 did not interact with the other PDZ domains of PSD95. Coexpression of G␥13 with its G1 partner did not interfere with these two-hybrid interactions. The physical interaction of G␥13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of G␥13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of G␥13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of G␥13 abolished its interactions with PSD95 in twohybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in G␥13-expressing taste cells. Coimmunoprecipitation of G␥13 and PSD95 from brain and of G␥13 and SAP97 from taste tissue indicates that G␥13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of G␥ subunits. The interaction of G␥13 with PDZ domain-containing proteins may provide a means to target particular G␥ subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.A sophisticated and ordered protein network is essential to the proper functioning of cells. Precise assembly of individual components, through targeting and anchoring of proteins within designated subcellular compartments, ensures the integrity of these networks (1, 2). Specific protein-protein interactions are important for accomplishing this complex task. For example, PSD95 (postsynaptic density protein 95, also called SAP90), a member of the MAGUK (membrane-associated guanylate kinase) protein family (3), helps to assemble a complex postsynaptic protein network via its interactions with several different proteins. Some of these interactions rely on three PDZ domains (named after PSD95, Disc-large, and ZO-1) located in the N-terminal half of PSD95. PDZ domains function as protein-protein interaction modules and consist of about 90 amino acids (4 -6).PDZ domains typically bind to the extreme C terminus of a target protein in a sequence-specific manner. The PDZ domains of PSD95 recognize a canonical -X(S/T)XA motif (where X represents any amino acid and A represents an aliphatic amino acid). PDZ domains have been identified in proteins in bacteria, yeast, Drosophila, metazoans, and plants and comprise the most common protein module identified in the sequenced genome (4, 6). In addition to their affinity for C-terminal motifs, PDZ domains can also bind to internal sequences that mimic free C termini. Finally, PDZ domains c...
Clustering analysis, as an important technique in data mining, aims to identify the nature groups or clusters of data objects in the attribute space. Data objects in real-world applications are commonly described by both numeric and categorical attributes. In this research, considering that the partitional clustering algorithms designed for this type of mixed data are prone to get trapped into local optima and the cuckoo search approach is efficient in solving global optimization problems, we propose CCS-K-Prototypes, a novel partitional Clustering algorithm based on Cuckoo Search and K-Prototypes, for clustering mixed numeric and categorical data. To deal with different types of attributes, we develop a novel representation for candidate solutions, and suggest two formulas for the cuckoo to search for the potential solution around the existing solutions or in the entire attribute space. Finally, the performance of the proposed algorithm is assessed by a series of experiments on five benchmark datasets. INDEX TERMS Data clustering, cuckoo search, mixed data, numeric and categorical attributes.
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