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Title: Biochemical and functional characterization of the RNA binding protein SAF-A/hnRNP U and investigation of its potential role in mammalian X chromosome inactivation
Author: Tattermusch, Anna
ISNI:       0000 0004 6062 3963
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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X chromosome inactivation (XCI) is an important mechanism for gene dosage compensation between XY males and XX females during early embryonic development of eutherian mammals. The major regulator of XCI is the long non-coding RNA (lncRNA) X inactive specific transcript (Xist). Xist RNA is believed to recruit epigenetic modifiers and initiate a silencing cascade that alters the chromatin structure and culminates in the transcriptional repression of most genes at the inactive X chromosome (Xi). Though extensively studied, the protein factors involved in the recruitment, spreading and maintenance of Xist RNA at the Xi are largely unknown. Recently published studies provide evidence that the DNA and RNA binding nuclear matrix protein scaffold attachment factor A/heterogeneous ribonucleoprotein U (SAF-A/hnRNP U) is required for Xist localization to the Xi. These studies, however, are incomplete in that they neither provide information on the RNA binding consensus sequence for SAF-A/hnRNP U on Xist, nor on its functional role in XCI. The present study was designed to re-investigate the role of SAF-A/hnRNP U in XCI using state-of-the-art techniques, notably fluorescence recovery after photo-bleaching (FRAP), 3D-structured illumination microscopy (3D-SIM), and in vivo crosslinking and RNA immunoprecipitation (CLIP). The results indicate that SAF-A/hnRNP U exists in two forms; a highly dynamic pan-nuclear protein and a stably Xi-associated form. 3D-SIM reveals that the latter closely associates with Xist RNA, and lines out the interchromatin channels within the Barr body. This finding suggests that SAF-A/hnRNP U takes part in the structural organization of the Xi by creating a secluded chromatin environment that retains XCI factors in their dedicated nuclear location. In contrast to observations made in microscopy imaging, biochemical experiments did not confirm a direct binding of SAF-A/hnRNP U to Xist RNA. This observation led to the conclusion that the protein plays an indirect, structural role in XCI.
Supervisor: Brockdorff, Neil Sponsor: Medical Research Council ; Stiftung der Deutschen Wirtschaft
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available