Alternative pre-mRNA splicing
of ion channels


WP1: Generation of new alternative exons through mobile genetic elements

WP1: Generation of new alternative exons through mobile genetic elements

Scientific team: Ida Vig, Noam Shomron, Galit Lev-Maor, Rotem Sorek, Noa Sela, Oren Ram, Amir Goren, Moti Ashkenazy, Guy Kol, Sharon Gazit, Gil Ast

Objectives: To determine the effect of exonized Alu elements and ChAB4 repeats on ion channel splicing in the nervous system. To examine features which distinguish beneficial exonization from those that can lead to genetic and/or acquired disorders.

Description of work: 99% of genes are highly conserved between human and mouse, which raises fundamental questions (e.g. what distinguishes human/primates from rodents). Retrotransposon elements (called Alu), are unique to primates. About 1.4 million Alu copies comprise more than 10% of the human genome. Recently, we demonstrated that more than 5% of the alternatively spliced human internal exons are Alu derived . Thus, this novel evolutionary pathway creates primate-specific genomic diversity. We propose to examine the exonization process by using a bioinformatics analysis complemented by experimental validation, subdivided into four aims:

  1. Compile a large dataset of exonized Alu (AEx).

  2. Sort this dataset for ion channels and their regulatory genes.

  3. Establish features that distinguish bona fide and non-functional alternatively spliced exons, and employ those features on each AEx.

  4. Test these predications in experimental systems.

Aim 1: Approximately 4 million human expressed sequence tags (ESTs) and cDNAs have been added to the public datasets during the past 2 years. Repeat of our bioinformatics analysis will therefore enlarge our dataset by additional mRNA isoforms containing AExs.
Aim 2
: To assess the roles played by the genes in the dataset, we will extract gene ontology (GO) annotations on these genes and identify those for receptors/ion channels, nervous system, and spliceosome component genes. All the GO terms for molecular function and biological processes will be extracted from the dataset and quantified.
Aim 3: Our recent comparison of mouse and human alternatively spliced exons identified conserved intronic elements that are probably involved in the regulation of alternative splicing. Additional distinguishing features will be identified using training exon sets to find a combination of features that detect alternative exons. We will use this system to examine each AEx and the human-specific ChAB4 repeats implicated in splice site regulation of trkB (WP5).
Aim 4
: Bioinformatic predictions will be tested experimentally in cell cultures. We already know four ion channels containing an AEx. Using brain tissue and cell lines, their splicing patterns will be examined by RT-PCR and immunohistochemistry, employing exon-specific antisera that will be raised against Alu-generated peptides. Minigenes containing/lacking an Alu element and a ChAB4 repeat will be tested in cotransfection assays. The mutated gene responsible for Familial Dysautonomia (FD), an autosomal recessive congenital neuropathy, contains an Alu element. We will test RNA from FD patient cells using the splicing regulator chip (WP6) to find putatively involved modifier sequences, transacting splicing factors and spliceosome genes.


Previous work related to the project:
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Sorek, R., R. Shamir, and G. Ast, How prevalent is functional alternative splicing in the human genome? Trends Genet, 2004. 20(2): p. 68-71.

Dagan, T., et al., AluGene: a database of Alu elements incorporated within protein-coding genes. Nucleic Acids Res, 2004. 32 Database issue: p. D489-92.

Lev-Maor, G., et al., The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons. Science, 2003. 300(5623): p. 1288-91.

Sorek, R. and G. Ast, Intronic sequences flanking alternatively spliced exons are conserved between human and mouse. Genome Res, 2003. 13(7): p. 1631-7.

Sorek, R., G. Ast, and D. Graur, Alu-containing exons are alternatively spliced. Genome Res, 2002. 12(7): p. 1060-7.