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Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Exploring the architecture of lncRNA RepA, a key player in X-chromosome inactivation

Recent work has demonstrated that long non-coding RNAs (lncRNAs) are essential components of the mammalian transcriptome (Flicek et al., 2014). They exhibit multiple structural and functional roles in various cellular processes and diseases (Wapinski and Chang, 2011; Sauvageau et al, 2013), and their analysis has become a new frontier in biomedical research. One of the most extensively studied lncRNAs is the X-inactivation specific transcript (Xist, 17 kb in mouse) (Brockdorff et al. 1991). During X-chromosome inactivation (XCI) in female mammals, Xist localizes to chromatin, spreads over the future inactive X-chromosome (Xi), and causes transcriptional silencing of X-linked genes (Brown and Willard, 1994; Csankovszki et al., 1999). It has been proposed that RepA, a lncRNA consisting of the same 1.6 kilobases as the 5' region of Xist, up-regulates the expression of Xist, and contributes to the initiation and spread of XCI (Zhao et al, 2008). Despite the availability of numerous functional data, little structural work has been performed to characterize the molecular mechanisms of RepA or Xist. In order to study its secondary structure, a non-denaturing purification protocol was employed to obtain a homogeneous and monodisperse form of RepA, and by studying its compaction with biophysical hydrodynamic methods, the ionic requirements for RepA folding were assessed. Chemical probing experiments (SHAPE and DMS probing) on the uniform, co-transcriptionally folded RNA sample, in parallel with phylogenetic analysis, revealed that RepA has a complex structural organization, with a variety of secondary structural motifs. It is comprised of three independently-folding modules, one of which is the 5' domain, which is a potential protein binding region whose secondary structure elements are found to be evolutionarily conserved. Our results provide the first secondary structural map of RepA, and offer structural insights for understanding the evolution and functional properties of this important lncRNA.

Reference
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    Brown, C.J. and Willard, H.F. (1994) The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature 368,154-156.

    Csankovszki, G., Panning, B., Bates, B., Pehrson, J.R., Jaenisch, R. (1999) Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat Genet. 22, 323-324.

    Flicek, P., Amode, M.R., Barrell, D., Beal, K., Billis, K., Brent, S., Carvalho-Silva, D., Clapham, P., Coates, G., Fitzgerald, S., et al. (2014). Ensembl 2014. Nucleic Acids Res 42, D749-D755.

    Sauvageau, M., Goff, L.A., Lodato, S., Bonev, B., Groff, A.F., Gerhardinger, C., Sanchez-Gomez, D.B., Hacisuleyman, E., Li, E., Spence, M., et al. (2013). Multiple knockout mouse models reveal lincRNAs are required for life and brain development. Elife 2, e01749.

    Wapinski, O., and Chang, H.Y. (2011). Long noncoding RNAs and human disease. Trends Cell Biol 21, 354-361.

    Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J., and Lee, J.T. (2008). Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750-756.


Fei Liu 1
Anna Marie Pyle1, 2, 3

1 Department of Molecular, Cellular and Developmental Biology
Yale University
New Haven, CT – 06511, USA
2 Department of Chemistry
Yale University
New Haven, CT – 06511, USA
3 Howard Hughes Medical Institute
Chevy Chase, MD – 20815, USA

Phone: (203) 432-5546
f.liu@yale.edu