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Goals and significance
The long-term aim of my
research is to understand the regulation and function of
alternative splicing. The sequencing of the human genome
has demonstrated the existence of only 22-35,000 genes,
far less than previously anticipated. Since the
transcriptome consists of at least 250,000 molecules,
pre-mRNA processing events in humans contribute more
significantly to human gene expression and regulation
than previously thought. Recent array data show that at
least 74% of all human genes are alternatively spliced.
Changes in alternative splice site selection are often
characteristic for developmental stages or certain cell
types, such as neurons or cells derived from the immune
system. Alternative splicing pathways are not static,
but can change according to environmental cues.
Therefore, alternative splicing emerges as one of the
most important mechanism to regulate gene expression. In
order to understand this regulatory function, I am
connecting results obtained by molecular biological
techniques and bioinformatics with physiological events.
Three major questions are being addressed:
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What is the mechanism
of alternative splicing, both genome-wide and in
well-studied model systems?
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How do signal
transduction pathways govern the use of
alternatively spliced exons?
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What is the mechanism
and a possible cure for changes in alternative
splicing in spinal muscular atrophy, tauopathies and
Prader-Willi Syndrome?
Mechanism of alternative splicing
Splice site selection is
regulated by the binding of trans-acting factors to RNA
sequence elements. These factors work in a concentration
dependent manner, which acts as a ‘cellular code’ that
needs to be deciphered. The challenge in determining the
regulation of splice sites is the high degeneracy of the
regulatory sequences and the low specificity of the
individual interactions. We therefore tackle this
problem from two ends: very detailed work in model
systems and genome-wide analysis. Both approaches
generate theories that can be tested by the other
method. To analyze alternative splicing on a genome-wide
level, we established databases of alternative exons,
their regulatory features and functions. Experimentally,
we characterized several new splicing regulatory
proteins and established model systems we can test both
in vivo and in vitro. Several of these model systems
(SMN2, tau and tra2-beta1) are relevant for human
diseases, i.e. spinal muscular atrophy, Alzheimer’s
disease and cancer. The advantage of the two-way
approach is illustrated by this example: We identified a
putative exonic enhancer in abnormal weak neuronal exons
by bioinformatic means. Later, we could demonstrate that
this enhancer is present in exon 10 of neurofilament tau,
where it binds to the protein tra2-beta1 that we
studied. A detailed analysis revealed that mutation of
this enhancer causes frontotemporal dementia and that
tra2-beta1 expression is changed in Alzheimer’s disease.
Signal transduction
pathways regulating alternative splicing
The use of alternative exons
often changes during development, or in response to
outside stimuli. However, the pathways that transduce
the signal to the splicing machinery remain to be
established. In order to develop therapeutic approaches
for diseases caused by wrong splice site selection, an
understanding of these signal transduction pathways is
necessary. We established several systems where we can
stimulate cells or intact animals and test changes in
splicing regulatory proteins. We found that
phosphorylation of splicing factors are at the end-point
of signal transduction pathways in all systems. The
phosphorylation of regulatory factors changes their
binding properties, resulting either in different RNP
complexes forming on the pre-mRNA or in sequestration of
splicing factors. As a result, splice site selection is
changed. Currently, we continue to analyze the
autoregulation of the tra2-beta system experimentally.
We found that the phosphorylation of tra2-beta1 is
regulated by protein phosphatase 1 and could demonstrate
that through this regulation alternative splicing events
are influenced through cAMP and cGMP levels that
regulate protein phosphatase 1 activity. These findings
let to novel drug candidates against spinal muscular
atrophy that are currently evaluated in mouse models.
Alternative (mis)splicing
and disease
Proper splicing regulation
is important for an organism. Point mutations in splice
sites cause an estimated 15% of genetic defects in
humans. Due to the growing awareness of the importance
of alternative splicing, this number has constantly
increased in the last years. We are investigating the
alternative splicing patterns of two genes, tau and SMN2
(survival of motoneuron 2) in more detail, because point
mutations in exonic enhancers in these genes result in
frontotemporal dementia with parkinsonism linked to
chromosome 17 (FTDP-17) and spinal muscular atrophy,
respectively. We could show that in both systems changes
in phosphorylation influence splice site selection,
which could be the basis of a novel therapeutic
approach. We found that tau exon 10 and its regulatory
factor tra2-beta1 is altered in Alzheimer’s disease.
This suggests that human diseases associated with
missplicing could be the result of a “wrong” combination
of regulatory factors. In an European consortium that I
coordinate, we have therefore developed an “alternative
exon chip” together with the necessary bioinformatic
tools. This allows us now to elucidate the cellular code
regulating splice site selection.
An important finding of the
human genome project is the abundance of small
non-coding RNAs. We were the first to demonstrate that
such small RNAs (snoRNAs) regulate alternative pre-mRNA
splicing. The lack of expression of one of this snoRNAs
causes Prader-Willi-syndrome by misregulating an
alternative splicing pattern of a serotonin receptor.
This represents a complete novel mechanism of
alternative splice site selection leading to a human
disease. |