Characterization of RNA interactions of dMi-2
Chromatin is maintained in a dynamic relaxed or repressed state such that DNA binding sites for the transcription machinery are either accessible or occluded. This way the gene expression is regulated. There are several chromatin regulators including ATP-dependent chromatin remodelers that help c...
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|Chromatin is maintained in a dynamic relaxed or repressed state such that DNA binding sites
for the transcription machinery are either accessible or occluded. This way the gene
expression is regulated. There are several chromatin regulators including ATP-dependent
chromatin remodelers that help change or modulate the conformation of the chromatin to
repress or to facilitate gene expression. Recent studies have implicated several chromatin
remodelers in RNA binding. However, the precise function of this RNA binding property is
intensively debated due to our limited understanding of its function. In this thesis, dMi-2, an
ATP-dependent chromatin remodeler, has been studied for its interaction with RNA and a
model is proposed for the effects of RNA binding on its function that supports the hypothesis
that RNA binding modulates the function of chromatin remodelers.
In the first part of this thesis, the RNA binding properties of dMi-2 were characterized in vitro.
dMi-2 formed biochemically stable complexes with several RNAs of varying sequences. This
suggested a binding of promiscuous nature. However, dMi-2 bound some RNAs with higher
affinity compared with others. Further, the RNA binding regions in dMi-2 were mapped. The
major RNA binding regions of dMi-2 were located in its N-terminal part. Analysis of the type
of sequences that dMi-2 bound to revealed a preference for G-rich RNA.
In the second part of this study, in vivo RNA binding of dMi-2 was characterized in an
unbiased genome-wide manner. The individual nucleotide-resolution crosslinking and
immunoprecipitation (iCLIP) experiment recapitulated the promiscuous nature of RNA
binding that was found in in vitro assays. iCLIP revealed thousands of different dMi-2-binding
RNAs. Further analysis revealed that dMi-2 interacted almost exclusively near the 3’-end of
the mRNAs. At the cross-linking site, G-nucleotides were enriched.
In the third part of this thesis, the effects of RNA binding on the function of dMi-2 were
studied. It was shown that RNA binding inhibits the remodeling activity of dMi-2. Further
analysis illustrated that the RNAs that bound dMi-2 with higher affinity also inhibited its
remodeling better compared with the RNAs that bound with lower affinity. Biochemical
analysis revealed that the depletion of RNA led to an increased chromatin occupancy of dMi-
2. Likewise, inhibition of transcription in vivo also resulted to an increased chromatin
occupancy of dMi-2.
In summary, the results of this thesis support a model which suggests that dMi-2-RNA
interaction causes inhibition of the remodeling function of dMi-2. This may be caused by
eviction of dMi-2 from chromatin upon RNA interaction. At actively transcribing genes, RNA
competes dMi-2 away from the chromatin and at repressed genes, dMi-2 has increased
chromatin occupancy due to lack of RNA. This thesis contributes to the understanding of the
role of RNA interaction in modulating the function of chromatin remodelers.