dc.description.abstract
Alternative splicing (AS) is a dynamic and highly regulated process to expand the
proteome diversity by allowing the generation of multiple possible messenger
RNA (mRNA) isoforms from a single primary transcript. The splicing process impacts
multiple biological processes, including gene expression (GE). By AS the cells can
rapidly adapt to changing internal or external stimuli, such as temperature changes. In
homeothermic organisms, the core body temperature oscillates in a circadian manner
in a range of around 1 °C - 4 °C and these subtle temperature changes are sufficient to
control AS. However, whether and how cells can sense and react to these minor changes
is not fully understood.
In this thesis, we identified the Cdc2-like kinases (CLKs) 1 and 4 as the temperature
sensors, which react to changes in the body temperature within a physiologically
relevant range with higher activity at lower temperatures. We showed that the mRNA
encoding the cold-inducible RNA binding protein (CIRBP) is CLK1/4-dependent
alternatively spliced at different temperatures, resulting in a warm-induced isoform
with lower stability.
Reversible phosphorylation-dependent mechanisms rely on the activity of
kinases and phosphatases. In contrast to CLK1/4, we showed the anti-oncogenic
protein phosphatase 2A (PP2A) to be more active at higher temperatures. RNA
sequencing (RNA-Seq) analyses revealed a global impact of PP2A on AS and GE
as phosphatase inhibition using okadaic acid (OA) almost completely abolished
temperature sensitivity in HEK293 cells. Besides, the tumor-suppressive transcription
factor p53 gets activated at higher temperatures in a PP2A-dependent manner, likely
through AS of MDM4. In contrast, oncogenic MYC is more active at lower temperatures,
which is consistent with negative regulation of phosphatase activity. These data
point to a novel, body temperature-dependent mechanism which activates p53
tumor-suppressive function and provides a molecular mechanism for the use of PP2A
inhibitors or hyperthermia in cancer therapies.
AS can result in the production of a premature translation termination codon (PTC)
and, thereby, control GE by nonsense-mediated mRNA decay (NMD). We found
for multiple RNA binding proteins (RBPs) that temperature-dependent alternatively
spliced isoforms are often targeted by the NMD pathway leading to body
temperature-responsive, rhythmic GE levels. As an example, we showed that the
temperature-dependent and NMD-inducing inclusion of SRSF10 exon 3 results in
reduced GE levels in a rhythmic manner and suggests that the production of the
NMD-targeted isoform is under control of an autoregulatory feedback loop.
In SRSF10, splicing of the NMD-inducing, exon 3-containing isoform is under
direct competition of a minor and a major splice site in SRSF10 exon 2. Finally,
we revealed that SRSF10 autoregulates its expression by activating the inclusion of
exon 3. Interestingly, usage of the minor splice site in exon 2, which leads to the
production of a coding mRNA, is reduced through the presence of a downstream
major splice site leading to AS of the NMD-inducing isoform. We found that SRSF10
transcript levels correlate with the minor spliceosome component RNA binding region
(RNP1, RRM) containing 3 (RNPC3) in a tissue- and developmental stage-specific
manner. Our data suggest that SRSF10 expression levels control the expression
of all other serine/arginine-rich (SR) proteins via cross-regulation. Therefore, the
minor spliceosome also controls major intron splicing, which globally affects AS and GE.
In summary, this work expands our knowledge of how cells sense and adapt to
changes in the body temperature. We showed that the activity of CLK1/4 and PP2A
is differently affected by fluctuations in the body temperature, resulting in distinctive
AS patterns and expression changes of target genes. We revealed that the temperature-dependent
AS-NMD pathway leads to cycling GE and identified the minor spliceosome
as a regulator of SRSF10 GE levels, which impacts the expression levels of all SR
proteins, thereby globally affecting major intron splicing.
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