Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1gamma gene

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U)GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U)GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1γ gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.     

Unexpectedly high allelic diversity at the KIT locus causing dominant white color in the domestic pig.

Mutations in KIT encoding the mast/stem cell growth factor receptor (MGF) are responsible for coat color variation in domestic pigs. The dominant white phenotype is caused by two mutations, a gene duplication and a splice mutation in one of the copies leading to skipping of exon 17. Here we applied minisequencing and pyrosequencing for quantitative analysis of the number of copies with the splice form. An unexpectedly high genetic diversity was revealed in white pigs. We found four different KIT alleles in a small sample of eight Large White females used as founder animals in a wild boar intercross. A similar number of KIT alleles was found in commercial populations of white Landrace and Large White pigs. We provide evidence for at least two new KIT alleles in pigs, both with a triplication of the gene. The results imply that KIT alleles with the duplication are genetically unstable and new alleles are most likely generated by unequal crossing over. This study provides an improved method for genotyping the complicated Dominant white/KIT locus in pigs. The results also suggest that some alleles may be associated with negative pleiotropic effects on other traits.     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.     

Whole-genome association study for the roan coat color in an intercrossed pig population between Landrace and Korean native pig

The roan coat color is characterized by white hairs intermingled with colored hairs. Candidate genes based on comparative phenotypes in horses and cattle involve the KIT and KIT ligand (MGF) genes. Here, we report the result of the whole genome scanning to detect genomic regions responsible for the roan coat color, using a three-generation pedigree of 62 pigs in an intercross between Landrace and Korean native pig. These pigs were genotyped using the PorcineSNP 60 BeadChip (Illumina, USA). The whole genome scan indicated that three genomic regions, 35～36 Mb, 38～39 Mb, and 58～59 Mb on SSC8, were commonly and highly associated/linked with the roan phenotype in the case/control, sib-pair, and linkage test, respectively. The porcine KIT was selected as a candidate gene, because it is located in one of the three significant regions and its function is related to coat color formation. SNPs and Indels within coding sequence (CDS), promoter, and 3′-UTR of KIT were surveyed. Twenty-two SNPs in the CDS reported previously, as well as nine variations in promoter (2 SNPs) and 3′-UTR (5 SNPs and 2 Indels) were detected. Although no causative mutations were identified, these results will help to elucidate the genetic mechanisms involved in the expression of the roan phenotype and will aid in identifying key mutations responsible for the roan phenotype in further studies.     

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3' splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3' splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3' splice sites.     

hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene

Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5′ splice site selection of exon 5 by interacting with its potential binding site close to 5′ splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5′ splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.     

HLA class II alleles in Ainu living in Hidaka district,Hokkaido, northern Japan

The Ainu people are considered to be the descendants of preagricultural native populations of northern Japan, while the majority of the population of contemporary Japan (Wajin) is descended mainly from postneolithic migrants. Polymorphisms of the HLA-DRB1, DRB3, and DQB1 alleles were investigated in DNA samples of 50 Ainu living in Hidaka district, Hokkaido. Unique features of the Ainu in this study were high incidences of DRB1*1401, DRB1*1406, and a newly described allele, DRB1*1106 (20%, 17%, and 5%, respectively). On the other hand, several common alleles in Wajin (DRB1*1502, 1302, 0803, and 1501) were found at relatively low frequencies (1–2%) in Ainu. Previously DRB1*1406 was described as a characteristic allele of some Native American or northeast Asian ethnic groups, and DRB1*1106 had been found in only two Singapore Chinese and one Korean. Principal component analysis of various populations based on HLA class II allele frequencies places the Ainu population midway between other east Asian populations, including Wajin, and Native Americans. These observations may support the hypothesis that the Ainu people are the descendants of some Upper Paleolithic populations of northeast Asia from which Native Americans are also descended. © 1996 Wiley-Liss, Inc.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.