WP4: Mobile genetic elements causing
hypertonic motor disorders through
missplicing of ion channels
Scientific
team: Kristina Becker, Andreas
Humeny, Marlen Braune, Julia Brill,
Silke Seeber,
Objectives: We use the
-subunit of the chloride channel
glycine receptor (GlyR) to identify
splicing factors as genetic modulators
of neuronal missplicing caused by mobile
genetic elements. Our aims are
-
Mapping of background genes
affecting murine hypertonic motor
phenotype by modulation of
missplicing of Glrb-alleles
-
Mass spectrometric analysis and
quantification of splice products
-
Characterization of splicing
modifier alleles in spastic
mice
-
Pathomechanisms of LINE1
retroelements in missplicing.
Description of the work:
GlyRs are ligand-gated chloride channels
mediating synaptic inhibition. Ligand
binding and ion channel properties are
determined by independently folding
domains of the GlyR subunit polypeptides
. GlyR subunits exist as splice
variants that are characterized by
alternatively spliced motifs within
the large cytoplasmic domain, giving
rise to functionally diverse ion
channels . Allelelic variants of the
human GlyR subunit genes GLRA1
and GLRB cause the hypertonic
motor disorder, hyperekplexia or
startle disease . Receptor and ion
channel mechanisms underlying motor
dysfunction have been studied in
phenotypically similar mouse mutants
(e.g., ). An intronic insertion of a
full-length LINE1 element into
the Glrb gene causes missplicing
of -subunit pre-mRNA in the mutant
mouse spastic, resulting in a
dramatic reduction, but not the complete
loss, of synaptic GlyRs. Reminiscent of
the hyperekplexia, in human patients
spastic mice from different
genetic backgrounds show profound
phenotypic variability. In C57BL/6J
mice, homozygosity for Glrbspa
is lethal. In contrast, B6C3Fe mice show
a mild phenotype corresponding to higher
levels of the correctly spliced
transcript. In a current genome-wide
analysis, we mapped this modulatory
effect on Glrbspa splice site
selection to a chromosomal position
near the locus of splicing factors of
the SR family. Modulation of -subunit
pre-mRNA splicing was confirmed by
cotransfection of one of these factors
(SFRS8, SWAP) with Glrbspa-minigenes
in cell culture experiments. In aim 1,
we will test candidate genes at the
modifier loci (Chr. 5, 72cM; Chr.11,
49cM) identified in our current studies,
then we will fine-map and resolve the
chromosomal loci associated with both,
the disease phenotype and the modulation
of missplicing by inserting the Glrbspa
allele into additional inbred lines
(e.g., CBA, BALBc), relying on
genotyping by
MALDI-TOF-MS
for informative SSLPs and/or SNPs.
In aim 2, the modifier effect
will be analyzed at the transcript level,
employing Chip-based techniques (WP6)
targeted at candidate genes of neuronal
signaling and pre-mRNA splicing.
Real-time PCR will be used to quantitate
of RT-RNA products in
different mouse phenotypes, complemented
by MALDI-TOF-MS based techniques
to establish the
correlation between a locus and the
disease phenotype. In aim 3, we will
functionally characterize the potential
splicing modifier. Strain-specific
differences in candidate gene function
(i.e. encoding splicing factors) will be
sought in cDNA microarray (WP6) and cDNA
sequencing. Strain-specific gene
variants of candidate spliceosome
components will be tested using in
ex-vivo-splicing assays in
strain-specific backgrounds. Finally,
the modulation of expression of
candidate splicing factors by external
factors, e.g. hormones or drugs, will be
tested in spastic mice of the different
phenotypic groups. In aim 4, we will
explore how the intronic retroelement
affects splicing. Using ex-vivo splicing
assays in strain- and genotype-specific
primary cell cultures, candidates
established by mapping will be
cotransfected with reporter genes that
contain or lack the LINE1 element.
Previous work related to the
project: |
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epileptic and ataxic Cacna2d2 mutant of
the mouse. J Biol Chem, 2004. 279(8):
p. 7322-30.
Breitinger, U., et al., Conserved
high affinity ligand binding and
membrane association in the native and
refolded extracellular domain of the
human glycine receptor alpha1-subunit.
J Biol Chem, 2004. 279(3): p.
1627-36.
Bonk, T., et al., Matrix-assisted
laser desorption/ionization
time-of-flight mass spectrometry-based
detection of microsatellite
instabilities in coding DNA sequences: a
novel approach to identify DNA-mismatch
repair-deficient cancer cells. Clin
Chem, 2003. 49(4): p. 552-61.
Accompanied by:
Comment: Petricoin, E.F. and L.A.
Liotta, Mass spectrometry-based
diagnostics: the upcoming revolution in
disease detection. Clin Chem, 2003.
49(4): p. 533-4.
Breitinger, H.G., et al., Opposing
effects of molecular volume and charge
at the hyperekplexia site alpha 1(P250)
govern glycine receptor activation and
desensitization. J Biol Chem, 2001.
276(32): p. 29657-63.
Saul, B., et al., Novel GLRA1
missense mutation (P250T) in dominant
hyperekplexia defines an intracellular
determinant of glycine receptor channel
gating. J Neurosci, 1999. 19(3):
p. 869-77.
Nikolic, Z., et al., The human
glycine receptor subunit alpha3. Glra3
gene structure, chromosomal
localization, and functional
characterization of alternative
transcripts. J Biol Chem, 1998.
273(31): p. 19708-14. |