Function of Satellite RNA Transcription for Centromere Assembly and Mitotic Spindle Formation
Final Report Abstract
The centromere is the fundamental higher-order structure in chromosomes that connects chromosomes to spindle microtubules in mitosis, and thus it is essential for their faithful segregation during cell division. Even though the centromeric regions have been viewed transcriptionally silent, few years ago it has been shown in several species that centromeric regions are transcribed and that the transcription or the produced centromeric RNAs participate in centromere function. However, the low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins, so the mechanism of action for centromeric RNAs remained unclear. The aim of this project was to obtain insight into the functional role of centromeric RNAs for kinetochore formation and how these non-coding RNAs participate to ensure fidelity of mitosis. First, using mouse embryonic stem cells, we showed that an imbalance in the levels of forward or reverse minor satellite transcripts, the centromeric RNAs in mouse, leads to severe chromosome segregation defects, and we found a feedback mechanism that leads to up-regulation of the opposite strand when we depleted the forward transcript, providing new insights into centromeric RNA homeostasis. Second, we developed a novel hypothesis for centromeric RNA function during the funding period. In our new concept, centromeric transcripts may function through secondary structure rather than sequence conservation. With our collaborators we showed that minor satellite RNA indeed adopts a stem-loop secondary structure, which is also conserved in human α-satellite transcripts. We identified an RNA binding region in one of the key kinetochore organizers, CENPC, and demonstrated that minor satellite transcripts bind to CENPC through the structured region of the RNA. Importantly, mutants that disrupt minor satellite secondary structure did not cause chromosome segregation defects in mouse embryonic stem cells, underlining that the structural motif, and not necessarily the underlying DNA sequence is key to centromeric RNA function. We therefore propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.
Publications
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Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells. Journal of Cell Biology, 223(7).
Chen, Yung-Li; Jones, Alisha N.; Crawford, Amy; Sattler, Michael; Ettinger, Andreas & Torres-Padilla, Maria-Elena
