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,
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:
-
Compile a large dataset of exonized
Alu (AEx).
-
Sort this dataset for ion channels
and their regulatory genes.
-
Establish features that distinguish
bona fide and non-functional
alternatively spliced exons, and
employ those features on each AEx.
-
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: |
|
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. |