Splice-site contribution in alternative splicing of PLP1 and DM20: molecular studies in oligodendrocytes

Mutations in the proteolipid protein 1 (PLP1) gene cause the X-linked dysmyelinating diseases Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia 2 (SPG2). We examined the severity of the following mutations that were suspected of affecting levels of PLP1 and DM20 RNA, the alternatively spliced products of PLP1: c.453G>A, c.453G>T, c.453G>C, c.453+2T>C, c.453+4A>G, c.347C>A, and c.453+28_+46del (the old nomenclature did not include the methionine codon: G450A, G450T, G450C, IVS3+2T>C, IVS3+4A>G, C344A, and IVS3+28-+46del). These mutations were evaluated by information theory-based analysis and compared with mRNA expression of the alternatively spliced products. The results are discussed relative to the clinical severity of disease. We conclude that the observed PLP1 and DM20 splicing patterns correlated well with predictions of information theory-based analysis, and that the relative strength of the PLP1 and DM20 donor splice sites plays an important role in PLP1 alternative splicing.     

Alteration of branch site consensus sequence and enhanced pre‐mRNA splicing of an NMDAR1 intron not associated with schizophrenia

Aberrant splicing of pre‐mRNA is recognized to account for a significant minority of disease‐causing mutations. The N‐methyl‐D ‐aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African‐American male near a weak putative branch‐site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in‐frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer‐extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African‐American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease. © 2002 Wiley‐Liss, Inc.     

Alteration of branch site consensus sequence and enhanced pre-mRNA splicing of an NMDAR1 intron not associated with schizophrenia

Aberrant splicing of pre-mRNA is recognized to account for a significant minority of disease-causing mutations. The N-methyl-D-aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African-American male near a weak putative branch-site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in-frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer-extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African-American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease.     

Control of the heat stress‐induced alternative splicing of a subset of genes by hnRNP K

Pre‐mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre‐mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA‐binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K‐dependent manner, whereas hnRNP K is not necessary for the stress‐induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA‐binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.     

SNPSplicer: systematic analysis of SNP-dependent splicing in genotyped cDNAs

Functional annotation of SNPs (as generated by HapMap (http://www.hapmap.org) for instance) is a major challenge. SNPs that lead to single amino acid substitutions, stop codons, or frameshift mutations can be readily interpreted, but these represent only a fraction of known SNPs. Many SNPs are located in sequences of splicing relevance-the canonical splice site consensus sequences, exonic and intronic splice enhancers or silencers (exonic splice enhancer [ESE], intronic splice enhancer [ISE], exonic splicing silencer [ESS], and intronic splicing silencer [ISS]), and others. We propose using sets of matching DNA and complementary DNA (cDNA) as a screening method to investigate the potential splice effects of SNPs in RT-PCR experiments with tissue material from genotyped sources. We have developed a software solution (SNPSplicer; http://www.ikmb.uni-kiel.de/snpsplicer) that aids in the rapid interpretation of such screening experiments. The utility of the approach is illustrated for SNPs affecting the donor splice sites (rs2076530:A>G, rs3816989:G>A) leading to the use of a cryptic splice site and exon skipping, respectively, and an exonic splice enhancer SNP (rs2274987:C/T), leading to inclusion of a new exon. We anticipate that this methodology may help in the functional annotation of SNPs in a more high-throughput fashion.     

The regulation and regulatory activities of alternative splicing of the SMN gene

Alternative splicing is an essential process that produces protein diversity in humans. It is also the cause of many complex diseases. Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder, is caused by the absence of or mutations in the Survival Motor Neuron 1 (SMN1) gene, which encodes an essential protein. A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 because exon 7 is alternatively spliced, producing a truncated protein, which is unstable. SMN1 and SMN2 differ by a critical C-to-T substitution at position 6 of exon 7 in SMN2 (C6U transition in mRNA). This substitution alone is enough to cause an exon 7 exclusion in SMN2. Various cis- and trans-acting factors have been shown to neutralize the inhibitory effects of C6U transition. Published reports propose models in which either abrogation of an enhancer element associated with SF2/ASF or gain of a silencer element associated with hnRNP A1 is the major cause of exon 7 exclusion in SMN2. Most recent model suggests the presence of an EXtended INhibitory ContexT (Exinct) that is formed as a consequence of C6U transition in exon 7 of SMN2. In Exinct model, several factors may affect exon 7 splicing through cooperative interactions. Such regulation may be common to many alternatively spliced exons in humans. Recent advances in our understanding of SMN gene splicing reveals multiple challenges that are specific to in vivo regulation, which we now know is intimately connected with other biological pathways.     

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene

In the rat beta-tropomyosin (beta-TM) gene, exons 6 and 7 are spliced alternatively in a mutually exclusive manner. Exon 6 is included in mRNA encoding nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle beta-TM. Previously, we demonstrated that a six nucleotide mutation at the 5' end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupting a putative secondary structure. The sequence in exon 7 acts as a bona fide exonic splicing silencer (ESS), which is bound specifically by a trans-acting factor. Isolation and peptide sequencing reveal that this factor is hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Binding of hnRNP H correlates with the ESS activity. Furthermore, addition of antibodies that specifically recognizes hnRNP H to the splicing reactions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP H. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue- and developmental-specific alternative splicing.     

A common polymorphism decreases low-density lipoprotein receptor exon 12 splicing efficiency and associates with increased cholesterol

Single nucleotide polymorphisms (SNPs) that alter exon splicing efficiency are an emerging class of functional genetic variants. Since mutations in low-density lipoprotein receptor (LDLR) are a primary cause of familial hypercholesterolemia, we evaluated whether LDLR SNPs may alter splicing efficiency and cholesterol homeostasis. A SNP within LDLR exon 12, rs688, was identified in silico as neutralizing a putative exon splicing enhancer. Studies in human liver samples established that this SNP was associated with significantly decreased LDLR exon 12 splicing efficiency in women in vivo. In vitro minigene splicing studies qualitatively replicated these in vivo results and demonstrated that rs688 specifically modulates splicing efficiency. These effects on splicing may be physiologically relevant because the presence of the rs688 minor allele associates with increased total and LDL-cholesterol in female members of the Framingham Offspring Study. The largest rs688-associated cholesterol differences were observed in pre-menopausal women. In summary, these studies identify an LDLR SNP present in approximately 60% of Caucasians that is associated with significant 10% increases in total and LDL-cholesterol in pre-menopausal women.     

STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.