Alternative pre-mRNA splicing
of ion channels


WP4: Mobile genetic elements causing hypertonic motor disorders through missplicing of ion channels

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, Cord-Michael Becker

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

  1. Mapping of background genes affecting murine hypertonic motor phenotype by modulation of missplicing of Glrb-alleles

  2. Mass spectrometric analysis and quantification of splice products

  3. Characterization of splicing modifier alleles in spastic mice

  4. 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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