The role and expression of the protocadherin-alpha clusters in the CNS

The clustered protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system. The family of clustered protocadherins (clustered Pcdh family) is substructured into three distinct gene arrays in mammals: Pcdh-alpha, Pcdh-beta, and Pcdh-gamma. These are regulated by multiple promoters and cis-alternative splicing without DNA recombination. Pcdh-alpha proteins interact with beta1-integrin to promote cell adhesion. They also form oligomers with Pcdh-gamma proteins at the same membrane sites. During neuronal maturation, Pcdh-alpha expression is dramatically downregulated by myelination. The clustered Pcdh family has multiple variable exons that differ somewhat in number and sequence across vertebrate species. At the single-cell level, Pcdh-alpha mRNAs are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons from each allele. These findings support the idea that diversified Pcdh molecules contribute to neural circuit development and provide individual cells with their specific identity.     

Functional significance of isoform diversification in the protocadherin gamma gene cluster

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.     

Mechanisms of Drosophila Dscam mutually exclusive splicing regulation

Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.     

PCDHA基因簇及其影响神经发育的研究进展

原钙黏连素(PCDHs)家族属于Ca2+依赖的细胞黏着糖蛋白,在脑神经元网络搭建中扮演至关重要的角色.PCDHs家族在染色体上呈现簇状和非簇状分布,簇内众多可变外显子在神经元内随机表达,其丰富的蛋白变体组合锚定在神经元表面,作为特有信号"密码",识别并介导轴突或树突之间的连接.该文综述了近些年国内外的研究报道,阐述家族...     

Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.     

Clustered protocadherin family

The brain is a complex system composed of enormous numbers of differentiated neurons, and brain structure and function differs among vertebrates. To examine the molecular mechanisms underlying brain structure and function, it is important to identify the molecules involved in generating neural diversity and organization. The clustered protocadherin (Pcdh) family is the largest subgroup of the diverse cadherin superfamily. The clustered Pcdh proteins are predominantly expressed in the brain and their gene structures in vertebrates are diversified. In mammals, the clustered Pcdh family consists of three gene clusters: Pcdh -α, Pcdh -β, and Pcdh -γ. During brain development, this family is upregulated by neuronal differentiation, and Pcdh-α is then dramatically downregulated by myelination. Clustered Pcdh expression continues in the olfactory bulb, hippocampus, and cerebellum until adulthood. Structural analysis of the first cadherin domain of the Pcdh-α protein revealed it lacks the features that classical cadherins require for homophilic adhesiveness, but it contains Pcdh-specific loop structures. In Pcdh-α, an RGD motif on a specific loop structure binds β1-integrin. For gene expression, the gene clusters are regulated by multiple promoters and alternative cis splicing. At the single-cell level, several dozen Pcdh -α and -γ mRNA are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons. The Pcdh-α and Pcdh-γ proteins also form oligomers, further increasing the molecular diversity at the cell surface. Thus, the unique features of the clustered Pcdh family may provide the molecular basis for generating individual cellular diversity and the complex neural circuitry of the brain.     

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.     

SNP-based large-scale identification of allele-specific gene expression in human B cells

Polymorphism and variations in gene expression provide the genetic basis for human variation. Allelic variation of gene expression, in particular, may play a crucial role in phenotypic variation and disease susceptibility. To identify genes with allelic expression in human cells, we genotyped genomic DNA and cDNA isolated from 31 immortalized B cell lines from three Centre d'Etude du Polymorphisme Humain (CEPH) families using high-density single-nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs. We identified seven SNPs in five genes with monoallelic expression, 146 SNPs in 125 genes with allelic imbalance in expression with preferentially higher expression of one allele in a heterozygous individual. The monoallelically expressed genes (ERAP2, MDGA1, LOC644422, SDCCAG3P1 and CLTCL1) were regulated by cis-acting, non-imprinted differential allelic control. In addition, all monoallelic gene expression patterns and allelic imbalances in gene expression in B cells were transmitted from parents to offspring in the pedigree, indicating genetic transmission of allelic gene expression. Furthermore, frequent allele substitution, probably due to RNA editing, was also observed in 21 genes in 23 SNPs as well as in 48 SNPs located in regions containing no known genes. In this study, we demonstrated that allelic gene expression is frequently observed in human B cells, and SNP chips are very useful tools for detecting allelic gene expression. Overall, our data provide a valuable framework for better understanding allelic gene expression in human B cells.     

Genetic analysis of four novel peroxisome proliferator activated receptor-gamma splice variants in monkey macrophages

Peroxisome proliferator activated receptor-gamma (PPAR-gamma) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-gamma 1, PPAR-gamma 2, and PPAR-gamma 3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-gamma exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5(') terminal region to generate four novel PPAR-gamma subtypes, PPAR-gamma 4, -gamma 5, -gamma 6, and -gamma 7. PPAR-gamma 4 and gamma 5 were detected only in macrophages whereas gamma 6 and gamma 7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-gamma exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-gamma 1, PPAR-gamma 4, and PPAR-gamma 5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-gamma expression in monkeys. RT-PCR showed a significant increase in total PPAR-gamma and ABCA1 mRNA levels in macrophages of fat-fed monkeys (n=7) compared to those maintained on a normal diet (n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-gamma 4 and -gamma 5 in CHO cells. In these cells, expression of PPAR-gamma 5 but not -gamma 4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-gamma isoforms may be responsible for unique tissue-specific biological effects and that PPAR-gamma 4 and -gamma 5 may modulate macrophage function and atherogenesis.