WP3: Regulation of splice site selection
of splicing factors
Scientific team: Ilona
Rafalska, Zhayi Zhang, Bettina Heinrich,
Shivendra Kishore, Natalya Benderskaya,
Tatyana Novoyatleva, Yesheng Tang,
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
interactions with other splicing
target RNAs, to determine
the influence of trkB-T1-activity
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
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
In aim 2, the target genes of the
splicing factors will be determined by
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
Previous work related to the
Stoilov, P., et al., Human tra2-beta1
autoregulates its protein concentration
by influencing alternative splicing of
its pre-mRNA. Hum Mol Genet, 2004.
13: p. 509-524.
Daoud, R., et al., Ischemia induces a
translocation of the splicing factor
tra2-beta1 and changes alternative
splicing patterns in the brain. J.
Neurosci., 2002. 22: p.
Stamm, S., Signals and their
transduction pathways regulating
alternative splicing: a new dimension of
the human genome. Hum. Mol. Genet.,
2002. 11: p. 2409-2416.
Hartmann, A.M., et al., Regulation of
alternative splicing of human tau exon
10 by phosphorylation of splicing
factors. Mol Cell Neurosci, 2001.
18(1): p. 80-90.
Daoud, R., et al., Activity-dependent
regulation of alternative splicing
patterns in the rat brain. Eur J
Neurosci, 1999. 11(3): p.