Alternative splicing of mutually exclusive exons—A review

Alternative splicing (AS) of pre-mRNAs in higher eukaryotes and several viruses is one major source of protein diversity. Usually, the following major subtypes of AS are distinguished: exon skipping, intron retention, and alternative 3′ and 5′ splice sites. Moreover, mutually exclusive exons (MXEs) represent a rare subtype. In the splicing of MXEs, two (or more) splicing events are not independent anymore, but are executed or disabled in a coordinated manner. In this review, several bioinformatics approaches for analyzing MXEs are presented and discussed. In particular, we revisit suitable definitions and nomenclatures, and bioinformatics tools for finding MXEs, adjacent and non-adjacent MXEs, clustered and grouped MXEs. Moreover, the molecular mechanisms for splicing MXEs proposed in the literature are reviewed and discussed.     

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA–protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model.     

RBM10 regulates alternative splicing

RBM10, originally called S1-1, is a nuclear RNA-binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis-related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl-x genes. RBM10 promotes exon skipping of Fas pre-mRNA as well as selection of an internal 5′-splice site in Bcl-x pre-mRNA. We propose a consensus RBM10-binding sequence at 5′-splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.     

mRNA选择性剪接的分子机制

真核细胞mRNA前体经过剪接成为成熟的mRNA,而mRNA前体的选择性剪接极大地增加了蛋白质的多样性和基因表达的复杂程度,剪接位点的识别可以以跨越内含子的机制(内含子限定)或跨越外显子的机制(外显子限定)进行。选择性剪接有多种剪接形式：选择不同的剪接位点,选择不同的剪接末端,外显子的不同组合及内含子的剪接与否等。选择性剪接过程受到许多顺式元件和反式因子的调控,并与基本剪接过程紧密联系,剪接体中的一些剪接因子也参与了对选择性剪接的调控。选择性剪接也是1个伴随转录发生的过程,不同的启动子可调控产生不同的剪接产物。mRNA的选择性剪接机制多种多样,已发现RNA编辑和反式剪接也可参与选择性剪接过程。     

Localization of DNA sequences governing alternative mRNA production of rat kininogen genes

The two types of the rat kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the one next to this exon (HMW exon) through alternative polyadenylation and splicing. In contrast, the two T genes selectively generate the LMW form of the mRNA, although the T genes are extremely homologous to the K gene, including the sequence (psi HMW region) corresponding to the HMW exon of the K gene. In this study, we constructed a series of chimeric kininogen genes by exchanging equivalent restriction fragments of the K and T genes and examined the sequences and the mechanisms governing the different expression patterns of the kininogen genes by introducing the chimeric genes into heterologous COS cells. The results indicate that the formation of the two forms of the mRNA is controlled by two separate 3' sequences of the kininogen genes. One is located within the internal sequence of the HMW/psi HMW region, whereas the other is within the LMW exon and its preceding region. Our data also suggest that the different expression patterns of the kininogen genes are primarily governed by differing splicing efficiency.     

Combinatorial control of exon recognition

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of noncoding intronic sequences to assemble exons into mature mRNAs prior to export and translation. Given the complexity of higher eukaryotic genes and the relatively low level of splice site conservation, the precision of the splicing machinery in recognizing and pairing splice sites is impressive. Introns ranging in size from <100 up to 100,000 bases are removed efficiently. At the same time, a large number of alternative splicing events are observed between different cell types, during development, or during other biological processes. This extensive alternative splicing implies a significant flexibility of the spliceosome to identify and process exons within a given pre-mRNA. To reach this flexibility, splice site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice site strength, the presence or absence of splicing regulators, RNA secondary structures, the exon/intron architecture, and the process of pre-mRNA synthesis itself. The relative contributions of each of these parameters control how efficiently splice sites are recognized and flanking introns are removed.     

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.