WP3: Regulation of splice site selection by Ca⁺⁺ concentration-dependent phosphorylation of splicing factors

Scientific team: Ilona Rafalska, Zhayi Zhang, Bettina Heinrich, Shivendra Kishore, Natalya Benderskaya, Tatyana Novoyatleva, Yesheng Tang, Stefan Stamm

Objectives: To

1. determine which Ca⁺⁺ dependent kinase(s) is responsible for the observed hyperphosphorylation of a subset of SR-proteins, especially TRA2-beta1, in brain ischemia, to identify the phosphorylation sites, to determine the phosphorylation-dependent interactions with other splicing factors and
2. target RNAs, to determine
3. the influence of trkB-T1-activity dependent Ca⁺⁺ fluctuations on alternative splicing.

Description of the work: Our previous work showed that several SR-proteins, among them tra2-beta1, are hyperphosphorylated after ischemia, and to a smaller extent following the neuronal activity burst during seizures. This hyperphosphorylation is caused by unknown Ca⁺⁺-dependent kinases after an increase in the intracellular calcium concentration and results in a change of splice site selection and the cytosolic accumulation of normally nuclear splicing factors.
In aim 1, Ca⁺⁺ dependent kinases will be identified by a) using commercially available panels of kinase inhibitors and b) cotransfection of the respective kinase(s). The hyperphosphorylation and cytosolic accumulation of EGFP-tagged splicing factors will be determined by PAGE and real-time fluorescent microscopy (WP5), respectively. Once the kinase is identified, GFP-tagged versions of splicing factors will be expressed in cells, isolated by immunoprecipitation and the phosphorylation sites will be determined by mass-spectroscopy (WP4). Using this information, mutants lacking phosphorylation properties (S,T->A, V; Y->F) or mimicking phosphorylation sites (A->E) will be constructed and phospho-specific antibodies generated. These mutant/wild type constructs will be transfected, immunoprecipitated and phosphorylation dependent interaction detected by mass-spectroscopy. In parallel, phosphorylation dependent interaction will be determined by the the yeast three-hybrid system, using the kinase to determine binding between SR-proteins and potentially novel interactors.
In aim 2, the target genes of the splicing factors will be determined by cross-linking and immunoprecipitation (CLIP) and compared with the CLIP results obtained from phosphorylated splicing factors. First, we will use over expression of GFP-tagged proteins for CLIP analysis. Then, we will use the endogenous proteins in primary neuronal cultures. Putative target genes identified by CLIP, preferentially ion channels will be cloned in frame with GFP reporters, resulting in splice site selection dependent GFP expression. These reporters will be tested in response to Ca⁺⁺ concentration changes, inhibition of Ca⁺⁺ dependent kinases and/or in cell lines lacking these kinases. In a parallel approach, we will directly test minigenes generated in WP1 containing the tra2-beta1 binding site GHVVGANR for their tra2-beta1 dependence. Calcium is a common second messenger, controlled by the acetylcholinesterase variant AChE-R through RACK1 (WP6) and released after BDNF binding to trkB-T1 (WP5).
In aim 3, we will use these systems to manipulate Ca⁺⁺ levels and determine their effect on tra2-beta, AChE-R and trk-B splice site selection, as well as on genes identified in WP1. These experiments will show how a Ca⁺⁺ signal changes the phosphorylation status of splicing regulatory proteins and which genes are affected by this regulation.
 

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