The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.     

Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae

In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC.     

A role for U2/U6 helix Ib in 5' splice site selection

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.     

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.     

Interaction between the negative regulator of splicing element and a 3' splice site: requirement for U1 small nuclear ribonucleoprotein and the 3' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.     

Genetic interaction between YPT6 and YPT1 in Saccharomyces cerevisiae

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. This study investigated the invasion of HeLa cells and its consequences by confocal laser scanning microscopy. Invasion was dependent on the duration of infection and temperature, diminished by inhibiting microfilament assembly with cytochalasin D and almost completely abolished by disorganising microtubules with vinblastine or taxol. After a short infection period (< 20 min), pronounced activation of protein kinase C was detected in host cells, whereas prolonged infection resulted in intensive vacuolation of the host cells and a pronounced increment in intracellular organic peroxide levels. A marked decrease in the extent of vacuolation was observed when peroxide accumulation was partially prevented by alpha-tocopherol. The possibility that M. penetrans entry into HeLa cells involves the activation of protein kinases and the recruitment of cytoskeleton components is discussed.     

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.     

A role for the actin cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection

The effects of structure on the estrogenicity and antiestrogenicity of hydroxylated polychlorinated biphenyls were investigated using the following estrogen-sensitive assays: competitive binding to the rat and mouse cytosolic estrogen receptor (ER); immature rat and mouse uterine wet weight, peroxidase and progesterone receptor (PR) levels; induction of luciferase activity in HeLa cells stably transfected with a Gal4:human ER chimera and a 17mer-regulated luciferase reporter gene; proliferation of MCF-7 human breast cancer cells; induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a CAT reporter gene. The chemicals synthesized for this study contained a 4-hydroxy group in one ring, a 2- or 3-chloro substituent meta or ortho to the hydroxyl group, and variable substitution (2',3',4',5'-, 2',3',4',6'-, 2',3',5',6'-tetrachloro and 2',4',6'-trichloro) in the chlorophenyl ring. The compounds included: 2,2',3',4',5'- (A), 2,2',3',4',6'- (B), and 2,2',3',5',6'-pentachloro- (C); 2,2',4',6'-tetrachloro-4-biphenylol (D); 2',3,3',4',5'- (E), 2',3,3',4',6'- (F), and 2',3,3',5',6'-pentachloro (G); and 2',3,4',6'-tetrachloro-4-biphenylol (H). With the exception of 2',3,4',6'-tetrachloro-4-biphenylol (H), all of the compounds competitively bound to the mouse and rat ER with relative binding affinities [compared to 17beta-estradiol (E2)] varying from 1.4 x 10(-3) to 5.3 x 10(-5). The structure-ER binding relationships for the hydroxy-PCB congeners were different in the rat and mouse, and no dose-dependent estrogenic activities were observed in the mouse or rat uterus. Several hydroxy-PCB congeners exhibited antiestrogenic activity (primarily in the mouse uterus) and two compounds, 2,2',3',5',6- and 2,2',3',4',6'-pentachloro-4-biphenylol, inhibited E2-induced uterine wet weight, PR binding, and peroxidase activity in the mouse uterus. 2,2',3',4',5'- and 2,2',3',4',6'-Pentachloro-4-biphenylol induced CAT activity in MCF-7 cells transiently transfected with the Vit-CAT plasmid; the remaining congeners did not induce CAT activity but exhibited antiestrogenic activity in MCF-7 cells cotreated with 10(-9) E2 plus 10(-5) M hydroxy-PCBs. Complementary structure-estrogenicity relationships were observed utilizing the HeLa cell luciferase induction and MCF-7 cell proliferation assays. The placement of the 2- or 3-chloro groups in the phenolic ring had minimal effects on estrogenic activity, whereas 2,4,6-trichloro- and 2,3,4,6-tetrachloro substitution in the chlorophenyl ring (B, D, F, and H) were required for this response. Substitution in the phenolic ring was also not important for structure-antiestrogenicity relationships, and the most active compounds (A, C, E, and G) contained 2',3',4',5'- and 2',3',5',6'-tetrachlorophenyl groups. Thus, structure-estrogenicity/antiestrogenicity relationships for this series of hydroxy-PCBs were complex and response-specific.     

Identification of exon sequences involved in splice site selection

The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons. When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct. Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals. A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold. Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold. Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection.     

Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron

Nuclear pre-messenger RNA splicing requires the action of five small nuclear (sn) RNAs, U1, U2, U4, U5 and U6, and more than 50 proteins. The mechanistic similarity of nuclear pre-mRNA splicing and group II self-splicing suggests that many of the central processes of nuclear pre-mRNA splicing are based on RNA-RNA interaction. To understand the mechanism of pre-mRNA splicing, the interactions, and their temporal relationships, that occur between the snRNAs and the pre-mRNA during splicing must be identified. Several snRNA-snRNA and snRNA-intron interactions have been demonstrated but the putative RNA-based interactions that recognize the AG dinucleotide at the 3' splice site during 3' cleavage and exon ligation are unknown. We report here the reciprocal suppression between 5' and 3' splice site mutations in the yeast actin intron, and propose that the 3' splice site is positioned for 3' cleavage and exon ligation, at least in part, through a non-Watson-Crick interaction between the guanosines at the 5' and 3' splice sites.     

Usage of cryptic splice sites in citrullinemia fibroblasts suggests role of polyadenylation in splice-site selection during terminal exon definition

Citrullinemia is a human genetic disease caused by a deficient argininosuccinate synthetase. In fibroblasts established from a citrullinemia patient with a mutation at the 3' splice site of the terminal intron of the gene, three cryptic 3' splice sites; i.e., SA1275, SA1636, and SA1663, residing on the terminal exon were activated. The usage of the cryptic sites showed a gradient, with the most downstream site having the highest usage; i.e., SA1663 > SA1636 > SA1275. However, when these cryptic sites were relocated to the internal exon, SA1636 was used the most. The splice-site strength of SA1636 was at least 10-fold higher than that of SA1663 in this situation. The results suggest that the preferential usage of SA1663 residing on the terminal exon may depend on its proximity to the poly(A) signal rather than on the strength of the splice site. Furthermore, when the strength of the downstream-most splice site increased, almost all the RNAs spliced to this site. However, in the presence of the wild-type splice site, all the RNAs were processed to the authentic site. Apparently, the selection of splice site can be revealed only when the sites being selected do not differ too much in their strength. By using a naturally occurring human mutant gene as a model, this study reveals that polyadenylation may play an important role in the selection of splice site during terminal exon definition.     

The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons

Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium [1]. More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis [2,3]. Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner. Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes. This may reflect differences in the functions of the actin cytoskeleton in these two organisms. Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S. cerevisiae [4,5] and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin [6]. We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton. The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles. Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant. Taken together, these results suggest that S. cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.     

Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

A cdc-like autolytic Saccharomyces cerevisiae mutant altered in budding site selection is complemented by SPO12, a sporulation gene

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lyt1-1 allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37 degrees C, lyt1 haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyt1-1/lyt1-1 diploids failed to sporulate at both 24 and 37 degrees C. Taking into account these characteristics, the lyt1 mutant could be considered a cdc-like mutant. By genetic transformation of an appropriate lyt1 strain with a genomic library, ligated to the multicopy vector YEp13, we isolated a gene capable of complementing mitotic alterations but not the meiotic defect. This was the sporulation-specific gene SPO12, which is expressed under the control of the locus MAT in meiosis and is also expressed in the mitotic cycle (V. Parkes and L. H. Johnston, Nucleic Acids Res. 20:5617-5623, 1992). A significant level of SPO12 mRNA can be detected when this gene is inserted in a multicopy plasmid.