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.     

U1 small nuclear ribonucleoprotein and splicing inhibition by the rous sarcoma virus negative regulator of splicing element

Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.     

The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly

Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3' splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3' splice site together act to inhibit splicing at the 3' splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3' splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3' splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.     

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.     

Cloning and characterization of PSF, a novel pre-mRNA splicing factor

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.     

An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.     

Bone structure of the temporo-mandibular joint in the individuals aged 18-25

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.     

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.     

HDL3-mediated inhibition of thrombin-induced platelet aggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate

We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.     

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.     

Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship

Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.     

Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin

A trimeric complex formed by Tub4p, the budding yeast gamma-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5' and 3' cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3' end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like gamma-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both gamma-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and gamma-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.     

Self-organization of binocular disparity tuning by reciprocal corticogeniculate interactions

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3' untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3' untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' exon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (> 36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. The 5' regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIA gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C-->A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2 + 11A/G, IVS4 + 44C/A, and IVS6 + 538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7 (CA)10-11 CCCCCTATA (TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene, HFE, were examined and found to be normal. One hemochromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromatosis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.     

Modulation of E-cadherin expression in TPA-induced cell motility: well-differentiated human adenocarcinoma cells move as coherent sheets associated with phosphorylation of E-cadherin-catenin complex

We have identified a human splicing factor required for the second step of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step. The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved. A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes. The homology identifies one region important for the function of both proteins and may define a new protein motif. In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.