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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Bibliographic Details
Main Author: Ullah, Ikram
Contributors: Brehm, Alexander (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2021
Subjects:
RNA
Online Access:PDF Full Text
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Summary: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.
Physical Description:220 Pages
DOI:https://doi.org/10.17192/z2021.0271