Conditional knockout mice to study alternative splicing in vivo

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease.     

Identifying novel genes in <Emphasis Type="Italic">C. elegans</Emphasis> using SAGE tags

Background  

Despite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes. This situation is partly explained by the fact that gene prediction programs have been developed based on our incomplete understanding of gene feature information such as splicing and promoter characteristics. Additionally, full-length cDNAs of many genes and their isoforms are hard to obtain due to their low level or rare expression. In order to obtain full-length sequences of all protein-coding genes, alternative approaches are required.     

Diversity of teleost leukocyte molecules: Role of alternative splicing

Alternative splicing is an important mechanism of gene expression control that also produces a large proteome from a limited number of genes. In the immune system of mammals, numerous relevant genes have been found to undergo alternative splicing that contributes to the complexity of immune response. An increasing number of reports have recently indicated that alternative splicing also occurs in other vertebrates, such as fish. In this review we summarize the general features of such molecular events in cytokines and leukocyte co-receptors and their contribution to diversity and regulation of fish leukocytes.     

中华猕猴桃基因组可变剪接事件鉴定及分析

可变剪接使一个基因能产生多种m RNA成熟体,极大地增加蛋白多样性.采用中华猕猴桃基因组数据做参考数据,利用中华猕猴桃叶片和果实3个不同发育时期(未成熟、半成熟和成熟期)的转录组数据,从中华猕猴桃基因组(39040个基因)中共鉴定出11651个基因(占总基因数的29%)对应的32180个可变剪接事件.在可变剪接不同类型中,内含子保留类型的发生频率最高,占50%以上;3′可变位点类型频率约为5′端可变类型的2倍.GO富集分析结果表明,可变剪接的基因主要富集于酶调控及核苷酸结合相关功能的GO类别中,而组织特有可变剪接基因功能富集热点与组织的重要功能关联,叶片多为肌动蛋白及微管相关;未成熟果实与双组分信号系统相关;半成熟果实多与磷脂合成过程相关;成熟果实多与信号传递过程相关.另外,55.6%的维生素合成相关基因发生可变剪接事件,显著高于基因组水平的29.6%,暗示着可变剪接参与维生素合成相关基因代谢过程中的重要作用.通过对中华猕猴桃全基因组可变剪接的分析,为解析中华猕猴桃基因组及进一步开展相关分子育种工作提供依据.     

The animal in the genome: comparative genomics and evolution

Comparisons between completely sequenced metazoan genomes have generally emphasized how similar their encoded protein content is, even when the comparison is between phyla. Given the manifest differences between phyla and, in particular, intuitive notions that some animals are more complex than others, this creates something of a paradox. Simplistic explanations have included arguments such as increased numbers of genes; greater numbers of protein products produced through alternative splicing; increased numbers of regulatory non-coding RNAs and increased complexity of the cis-regulatory code. An obvious value of complete genome sequences lies in their ability to provide us with inventories of such components. I examine progress being made in linking genome content to the pattern of animal evolution, and argue that the gap between genomic and phenotypic complexity can only be understood through the totality of interacting components.     

Regulation of splicing by SR proteins and SR protein-specific kinases

Genomic sequencing reveals similar but limited numbers of protein-coding genes in different genomes, which begs the question of how organismal diversities are generated. Alternative pre-mRNA splicing, a widespread phenomenon in higher eukaryotic genomes, is thought to provide a mechanism to increase the complexity of the proteome and introduce additional layers for regulating gene expression in different cell types and during development. Among a large number of factors implicated in the splicing regulation are the SR protein family of splicing factors and SR protein-specific kinases. Here, we summarize the rules for SR proteins to function as splicing regulators, which depend on where they bind in exons versus intronic regions, on alternative exons versus flanking competing exons, and on cooperative as well as competitive binding between different SR protein family members on many of those locations. We review the importance of cycles of SR protein phosphorylation/dephosphorylation in the splicing reaction with emphasis on the recent molecular insight into the role of SR protein phosphorylation in early steps of spliceosome assembly. Finally, we highlight recent discoveries of SR protein-specific kinases in transducing growth signals to regulate alternative splicing in the nucleus and the connection of both SR proteins and SR protein kinases to human diseases, particularly cancer.     

Origin and evolution of spliceosomal introns

ABSTRACT: Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers' Reports section.     

Functional impact of transposable elements using bioinformatic analysis and a comparative genomic approach

A dual coding event, which is the translation of different isoforms from a single gene, is one of the special patterns among the alternative splicing events. This is an important mechanism for the regulation of protein diversity in human and mouse genomes. Although the regulation for dual coding events has been characterized in a few genes, the individual mechanism remains unclear. Numerous studies have described the exonization of transposable elements, which is the splicing mediated insertion of transposable element sequence fragments into mature mRNAs. Therefore, in this study, we investigated the number of transposable element (TE)-derived dual coding genes in human, chimpanzee and mouse genomes. TE fusion exons appeared in the dual coding regions of 309 human genes. Functional protein domain alterations by TE-derived dual coding events were observed in 129 human genes. Comparative TE-derived dual coding events were also analyzed in chimpanzee and mouse orthologs. Seventy chimpanzee orthologs had TE-derived dual coding events, but mouse orthologs did not have any TE-derived dual coding events. Taken together, our analyses listed the number of TE-derived dual coding genes which could be investigated by experimental analysis and suggested that TE-derived dual coding events were major sources for the functional diversity of human genes, but not mouse genes.