%0 Generic
%A Dolejs, Vojtech
%C Heidelberg
%D 2024
%F heidok:35344
%R 10.11588/heidok.00035344
%T Protein-RNA interactions in centromeric chromatin
%U https://archiv.ub.uni-heidelberg.de/volltextserver/35344/
%X Chromosome segregation is a complex and tightly regulated process that is necessary for   proper cell division. Chromosomes are aligned at the equatorial plate and transported to   nascent daughter cells using motor proteins and microtubules. They are attached to the   microtubules by a multiprotein complex, the kinetochore. Kinetochores are anchored to  the centromeric regions of the chromosome. These regions consist of repetitive noncoding   DNA which forms the centromeric heterochromatin and is embedded in larger regions of   noncoding pericentromeric heterochromatin. Centromeres are not defined by an   underlying DNA sequence, but rather by the epigenetic marker Cenp-A, a histone H3   variant specific for centromeres.   Cenp-A, or Cid in Drosophila melanogaster (fruit fly), together with its loading factor   Cal1 and the Cenp-C protein form the basis of the inner kinetochore complex. The outer   kinetochore then connects to the spindle microtubule. This process requires the presence   of noncoding RNAs. These RNAs are transcribed from the pericentromeric regions and   colocalise with the inner parts of the kinetochore. The absence of these RNAs leads to   chromosome segregation defects, but their exact function as well as their interaction   partners are not well described. These defects are observed not just in Drosophila   melanogaster, but also in every other organism that has been researched to date.   In this work, I investigated the interaction of centromeric proteins of Drosophila   melanogaster with centromeric RNA using biochemical assays (EMSA) and mass   spectrometry (HDX-MS). I overexpressed and purified proteins as well as in vitro  transcribed RNAs. I observed the interaction of kinetochore proteins and centromeric   RNA with focus on Cal1 and satellite RNA. I demonstrated that Cal1 fragments are   RNA-binding using EMSA. However, HDX-MS was not sufficient to measure the details   of the interaction, as addition of RNA did not significantly change the deuteration pattern   of the protein fragments. I observed that Cal1 is unfolded and the fragment I worked with   is a promiscuous RNA-binder.