Computational identification of tissue-specific alternative splicing elements in mouse genes from RNA-Seq

Tissue-specific alternative splicing is a key mechanism for generating tissue-specific proteomic diversity in eukaryotes. Splicing regulatory elements (SREs) in pre-mature messenger RNA play a very important role in regulating alternative splicing. In this article, we use mouse RNA-Seq data to determine a positive data set where SREs are over-represented and a reliable negative data set where the same SREs are most likely under-represented for a specific tissue and then employ a powerful discriminative approach to identify SREs. We identified 456 putative splicing enhancers or silencers, of which 221 were predicted to be tissue-specific. Most of our tissue-specific SREs are likely different from constitutive SREs, since only 18% of our exonic splicing enhancers (ESEs) are contained in constitutive RESCUE-ESEs. A relatively small portion (20%) of our SREs is included in tissue-specific SREs in human identified in two recent studies. In the analysis of position distribution of SREs, we found that a dozen of SREs were biased to a specific region. We also identified two very interesting SREs that can function as an enhancer in one tissue but a silencer in another tissue from the same intronic region. These findings provide insight into the mechanism of tissue-specific alternative splicing and give a set of valuable putative SREs for further experimental investigations.     

Epigenetics in alternative pre-mRNA splicing

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.     

Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.     

Regulation of apoptosis by alternative pre-mRNA splicing

Apoptosis, a phenomenon that allows the regulated destruction and disposal of damaged or unwanted cells, is common to many cellular processes in multicellular organisms. In humans more than 200 proteins are involved in apoptosis, many of which are dysregulated or defective in human diseases including cancer. A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor. The abundance of apoptosis genes that are alternatively spliced and the often antagonistic roles of the generated protein isoforms strongly imply that alternative splicing is a crucial mechanism for regulating life and death decisions. Importantly, modulation of isoform production of cell death proteins via pharmaceutical manipulation of alternative splicing may open up new therapeutic avenues for the treatment of disease.     

Integrity of the exon 6 sequence is essential for tissue-specific alternative splicing of human leukocyte common antigen pre-mRNA.

By alternative splicing, exons 4, 5, and 6 of the human leukocyte common antigen (LCA) gene are included in B-cell mRNA but excluded from thymocyte mRNA. A mini-LCA gene that contains only LCA exons 2, 6, and 8 faithfully reproduces this tissue-specific alternative splicing in mouse B and thymocyte cell lines. Elimination of almost all of the intron sequences associated with exon 6 had no effect on the alternative splicing, while linker-scanning analysis showed that a significant length of the exon 6 sequence is essential for alternative splicing.     

Computational analysis of alternative splicing using EST tissue information

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.     

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis

Background

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.

Results

To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.

Conclusions

Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.     

Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.