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A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

ABSTRACT: Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40-60%), whereas the frequency of HPRT splice variants is considerably lower (approximately 1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed.

SUBMITTER: Skandalis A

PROVIDER: S-EPMC545452 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

Skandalis Adonis A Uribe Elke E

Nucleic acids research 20041215 22

Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcrip ...[more]

PMID: 15601998

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Project description:Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in Lesch-Nyhan disease (LND), where affected individuals exhibit a characteristic neurobehavioral disorder that has been linked with dysfunction of dopaminergic pathways of the basal ganglia. Since the functions of HPRT, a housekeeping enzyme responsible for recycling purines, have no direct relationships with the dopaminergic pathways, the mechanisms whereby HPRT deficiency affect them remain unknown. The current studies demonstrate that HPRT deficiency influences early developmental processes controlling the dopaminergic phenotype, using several different cell models for HPRT deficiency. Microarray methods and quantitative PCR were applied to 10 different HPRT-deficient (HPRT(-)) sublines derived from the MN9D cell line. Despite the variation inherent in such mutant sublines, several consistent abnormalities were evident. Most notable were increases in the mRNAs for engrailed 1 and 2, transcription factors known to play a key role in the specification and survival of dopamine neurons. The increases in mRNAs were accompanied by increases in engrailed proteins, and restoration of HPRT reverted engrailed expression towards normal levels, demonstrating a functional relationship between HPRT and engrailed. The functional relevance of the abnormal developmental molecular signature of the HPRT(-) MN9D cells was evident in impoverished neurite outgrowth when the cells were forced to differentiate chemically. To verify that these abnormalities were not idiosyncratic to the MN9D line, HPRT(-) sublines from the SK-N-BE(2) M17 human neuroblastoma line were evaluated and an increased expression of engrailed mRNAs was also seen. Over-expression of engrailed occurred even in primary fibroblasts from patients with LND in a manner that suggested a correlation with disease severity. These results provide novel evidence that HPRT deficiency may affect dopaminergic neurons by influencing early developmental mechanisms.

Project description:IntroductionExtracellular/environmental stimuli trigger cellular responses to allow Schistosoma sp. parasites adaptation and decide development and survival fate. In this context, signal transduction involving eukaryotic protein kinases (ePKs) has an essential role in regulatory mechanisms. Functional studies had shown the importance of MAPK pathway for Schistosoma mansoni development. In addition, early studies demonstrated that Smp38 MAPK regulates the expression of a large set of genes, among them the hypoxanthine-guanine phosphoribosyl transferase 1 (SmHGPRTase 1, Smp_103560), a key enzyme in the purine salvage pathway that is part of a family comprising five different proteins.MethodsFirst, the regulation of this gene family by the MAPKs pathways was experimentally verified using Smp38-predicted specific inhibitors. In silico analysis showed significant differences in the predicted structure and the domain sequence among the schistosomal HGPRTase family and their orthologs in humans. In order to interrogate the HGPRTases (Smp_103560, Smp_148820, Smp_168500, Smp_312580 and Smp_332640, henceforth SmHGPRTase -1, -2, -3, -4, -5) functional roles, schistosomula, sporocysts, and adult worms were knocked-down using specific dsRNAs.ResultsOur results suggest that SmHGPRTases activity has an essential role in sporocysts and schistosomula development since significant differences in viability, size, and/ or shape were observed after the in vitro knockdown. Also, the knockdown of SmHGPRTases in schistosomula influenced the ovary development and egg maturation in female adult worms during mammalian infection. We also observed alterations in the movement of female adult worms knocked-down in vitro. Most of these results were shown when all gene family members were knocked-down simultaneously, suggesting a redundant function among them.DiscussionThus, this study helps to elucidate the functional roles of the SmHGPRTase gene family in the S. mansoni life cycle and provides knowledge for future studies required for schistosomiasis treatment and control.

Project description:BACKGROUND: Alternative splicing (AS) is a key molecular process that endows biological functions with diversity and complexity. Generally, functional redundancy leads to the generation of new functions through relaxation of selective pressure in evolution, as exemplified by duplicated genes. It is also known that alternatively spliced exons (ASEs) are subject to relaxed selective pressure. Within consensus sequences at the splice junctions, the most conserved sites are dinucleotides at both ends of introns (splice dinucleotides). However, a small number of single nucleotide polymorphisms (SNPs) occur at splice dinucleotides. An intriguing question relating to the evolution of AS diversity is whether mutations at splice dinucleotides are maintained as polymorphisms and produce diversity in splice patterns within the human population. We therefore surveyed validated SNPs in the database dbSNP located at splice dinucleotides of all human genes that are defined by the H-Invitational Database. RESULTS: We found 212 validated SNPs at splice dinucleotides (sdSNPs); these were confirmed to be consistent with the GT-AG rule at either allele. Moreover, 53 of them were observed to neighbor ASEs (AE dinucleotides). No significant differences were observed between sdSNPs at AE dinucleotides and those at constitutive exons (CE dinucleotides) in SNP properties including average heterozygosity, SNP density, ratio of predicted alleles consistent with the GT-AG rule, and scores of splice sites formed with the predicted allele. We also found that the proportion of non-conserved exons was higher for exons with sdSNPs than for other exons. CONCLUSIONS: sdSNPs are found at CE dinucleotides in addition to those at AE dinucleotides, suggesting two possibilities. First, sdSNPs at CE dinucleotides may be robust against sdSNPs because of unknown mechanisms. Second, similar to sdSNPs at AE dinucleotides, those at CE dinucleotides cause differences in AS patterns because of the arbitrariness in the classification of exons into alternative and constitutive type that varies according to the dataset. Taking into account the absence of differences in sdSNP properties between those at AE and CE dinucleotides, the increased proportion of non-conserved exons found in exons flanked by sdSNPs suggests the hypothesis that sdSNPs are maintained at the splice dinucleotides of newly generated exons at which negative selection pressure is relaxed.

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A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

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A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

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