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.     

Distribution of exonic splicing enhancer elements in human genes

Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences.     

Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo

The U5 snRNA loop 1 is characterized by the conserved sequence G₁C₂C₃U₄U₅U₆Y₇A₈Y₉ and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae. Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro. To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted. Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation. These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo. A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1. Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns. The cell cycle arrest is likely attributed to inefficient splicing of α-tubulin pre-mRNA in one mutant and actin pre-mRNA in another. These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo.     

Mutations in apoptosis genes: a pathogenetic factor for human disease

Cell death by apoptosis is exerted by the coordinated action of many different gene products. Mutations in some of them, acting at different levels in the apoptosis process, have been identified as cause or contributing factor for human diseases. Defects in the transmembrane tumor necrosis factor receptor 1 (TNF-R1) lead to the development of familial periodic fever syndromes. Mutations in the homologous receptor Fas (also named CD95; Apo-1) are observed in malignant lymphomas, solid tumors and the autoimmune lymphoproliferative syndrome type I (ALPS I). A mutation in the ligand for Fas (Fas ligand; CD95 ligand, Apo-1 ligand), which induces apoptosis upon binding to Fas, was described in a patient with systemic lupus erythematodes and lymphadenopathy. Perforin, an other cytotoxic protein employed by T- and NK-cells for target cell killing, is mutated in chromosome 10 linked cases of familial hemophagocytic lymphohistiocytosis. Caspase 10, a representative of the caspase family of proteases, which plays a central role in the execution of apoptosis, is defect in autoimmune lymphoproliferative syndrome type II (ALPS II). The intracellular pro-apoptotic molecule bcl-10 is frequently mutated in mucosa-associated lymphoid tissue (MALT) lymphomas and various non-hematologic malignancies. The p53, an executioner of DNA damage triggered apoptosis, and Bax, a pro-apoptotic molecule with the ability to perturb mitochondrial membrane integrity, are frequently mutated in malignant neoplasms. Anti-apoptotic proteins like bcl-2, cellular-inhibitor of apoptosis protein 2 (c-IAP2) and neuronal apoptosis inhibitory protein 1 (NAIP1) are often altered in follicular lymphomas, MALT lymphomas and spinal muscular atrophy (SMA), respectively. This article reviews the current knowledge on mutations of apoptosis genes involved in the pathogenesis of human diseases and summarises the gradual transformation of discoveries in apoptosis research into benefits for the clinical management of diseases.     

Mutations in cardiovascular connexin genes

Connexins (Cxs) form a family of transmembrane proteins comprising 21 members in humans. Cxs differ in their expression patterns, biophysical properties and ability to combine into homomeric or heteromeric gap junction channels between neighbouring cells. The permeation of ions and small metabolites through gap junction channels or hemichannels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. Among others, Cx37, Cx40, Cx43, Cx45 and Cx47 are found in heart, blood and lymphatic vessels. Mutations or polymorphisms in the genes coding for these Cxs have not only been implicated in cardiovascular pathologies but also in a variety of other disorders. While mutations in Cx43 are mostly linked to oculodentodigital dysplasia, Cx47 mutations are associated with Pelizaeus–Merzbacher‐like disease and lymphoedema. Cx40 mutations are principally linked to atrial fibrillation. Mutations in Cx37 have not yet been described, but polymorphisms in the Cx37 gene have been implicated in the development of arterial disease. This review addresses current knowledge on gene mutations in cardiovascular Cxs systematically and links them to alterations in channel properties and disease.     

Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes

Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing.     

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.