Defective splicing induced by 4NQO in the hamster hprt gene

The molecular analysis of mutations affecting mRNA processing may contribute to a better understanding of the splicing mechanism through the identification of genomic sequences necessary for the recognition of splice sites. In this paper we report the sequence analysis of 14 splice mutants induced by 4-nitroquinoline 1-oxide (4NQO) at the hamster hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus. We show that mutations at the 3′ acceptor splice site or at the first or fifth base of the 5′ donor splice site are responsible for exon skipping. In addition, mutations in exon sequences also determine the skipping of one or more exons. Our data indicate that point mutations in intron regions at either side of an internal exon may induce the skipping of the same exon, supporting a model where the exon is the unit of early spliceosome assembly. Furthermore, they suggest that the splicing of hprt mRNA precursors may proceed through a clustering of exons 2, 3 and 4 which are then spliced in a concerted way.     

Genetic and functional analysis of the DLG4 gene encoding the post-synaptic density protein 95 in schizophrenia

Hypofunction of N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction has been implicated in the pathophysiology of schizophrenia. Post-synaptic density protein 95 (PSD95) plays a critical role in regulating the trafficking and activity of the NMDA receptor and altered expression of the PSD95 has been detected in the post-mortem brain of patients with schizophrenia. The study aimed to examine whether the DLG4 gene that encodes the PSD95 may confer genetic susceptibility to schizophrenia. We re-sequenced the core promoter, all the exons, and 3′ untranslated regions (UTR) of the DLG4 gene in 588 Taiwanese schizophrenic patients and conducted an association study with 539 non-psychotic subjects. We did not detect any rare mutations at the protein-coding sequences of the DLG4 gene associated with schizophrenia. Nevertheless, we identified four polymorphic markers at the core promoter and 5′ UTR and one single nucleotide polymorphism (SNP) at the 3′UTR of the DLG4 gene in this sample. Genetic analysis showed an association of a haplotype (C–D) derived from 2 polymorphic markers at the core promoter (odds ratio = 1.26, 95% confidence interval = 1.06–1.51, p = 0.01), and a borderline association of the T allele of the rs13331 at 3′UTR with schizophrenia (odds ratio = 1.19, 95% confidence interval = 0.99–1.43, p = 0.06). Further reporter gene assay showed that the C-D-C-C and the T allele of the rs13331 had significant lower activity than their counter parts. Our data indicate that the expression of the DLG4 gene is subject to regulation by the polymorphic markers at the core promoter region, 5′ and 3′UTR of the gene, and is associated with the susceptibility of schizophrenia.     

Multiple features contribute to efficient constitutive splicing of an unusually large exon

Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5′ splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5′ splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5′ splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.     

Myosin light-chain 1/3 gene alternative splicing: cis regulation is based upon a hierarchical compatibility between splice sites.

The mechanisms involved in the selective joining of appropriate 5' and 3' splice sites are still poorly understood in both constitutive and alternatively spliced genes. With two promoters associated with different exons, the myosin light-chain 1/3 gene generates two pre-mRNAs that also differ by the use of a pair of internal exons, 3 and 4, that are spliced in a mutually exclusive fashion. When the promoter upstream from exon 1 is used, only exon 4 is included. If the promoter upstream from exon 2 is used, only exon 3 is included. In an attempt to understand the molecular basis for the mutually exclusive behavior of these two exons and the basis of their specific selection, a number of minigene constructs containing exons 3 and 4 were tested in a variety of homologous or heterologous cis and trans environments. The results demonstrate that the mutually exclusive behavior of myosin light-chain exons 3 and 4 and selection between the two exons are cis regulated and are affected by the nature of the flanking sequences. Both exons competed for the common flanking 5' and 3' splice sites. Flanking exons were found that favored inclusion into mature mRNA of exon 3, exon 4, both, or neither, suggesting a specific cooperative interaction between certain 5' and 3' splice sites. Thus, alternative splicing of myosin light-chain 1/3 pre-mRNAs is regulated in cis by a hierarchy of compatibilities between pairs of 5' and 3' splice sites.     

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences.

Mutations resulting in defective splicing constitute a significant proportion (30/62 [48%]) of a new series of mutations in the ATM gene in patients with ataxia-telangiectasia (AT) that were detected by the protein-truncation assay followed by sequence analysis of genomic DNA. Fewer than half of the splicing mutations involved the canonical AG splice-acceptor site or GT splice-donor site. A higher percentage of mutations occurred at less stringently conserved sites, including silent mutations of the last nucleotide of exons, mutations in nucleotides other than the conserved AG and GT in the consensus splice sites, and creation of splice-acceptor or splice-donor sites in either introns or exons. These splicing mutations led to a variety of consequences, including exon skipping and, to a lesser degree, intron retention, activation of cryptic splice sites, or creation of new splice sites. In addition, 5 of 12 nonsense mutations and 1 missense mutation were associated with deletion in the cDNA of the exons in which the mutations occurred. No ATM protein was detected by western blotting in any AT cell line in which splicing mutations were identified. Several cases of exon skipping in both normal controls and patients for whom no underlying defect could be found in genomic DNA were also observed, suggesting caution in the interpretation of exon deletions observed in ATM cDNA when there is no accompanying identification of genomic mutations.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.