Talk on Saturday 03:00-03:12pm submitted by Yizhu Lin
High-throughput probing of human lncRNA secondary structure by Mod-seq
Yizhu Lin (Department of Biological Sciences, Carnegie Mellon University), Gemma E. May (Department of Biological Sciences, Carnegie Mellon University), C. Joel McManus (Department of Biological Sciences, Carnegie Mellon University)
Over the past decade, long noncoding RNAs (lncRNAs) have been increasingly recognized as important regulators of multiple gene expression processes. A growing number have been associated with human development and diseases. One recently discovered lncRNA family, sno-lncRNA, are transcribed from a region responsible for two human neurological diseases, Prader-Willi and Angelman Syndromes (Yin et al., 2012). sno-lncRNAs are so named for their genomic architecture – they contain box C/D or box H/ACA snoRNAs at both their 5’ and 3’ ends. Yin and colleagues have shown that the Rbfox splicing factor Fox2 binds to sno-lncRNAs and that sno-lncRNA expression changes the alternative splicing of Fox2 targets. Because of their unique architecture and potential role as Fox2-binding sponges, understanding the biological function of sno-lncRNAs requires characterization of their structure.
To experimentally determine the secondary structures of sno-lncRNAs, we applied a recently developed method, Mod-seq (Talkish et al., 2014). Mod-seq allowed the rapid identification of SHAPE (1M7) modifications on the full length (1,178 nt) sno-lncRNA2. By calibrating our Mod-seq results with those produce by SHAPE probing of the P4-P5 domain of the tetrahymena group I intron, we calculated SHAPE-scores and used RNAstructure to produce models of sno-lncRNA2 secondary structure. The SHAPE constrained secondary structure of sno-lncRNA2 shows only ~36% similarity with the structure predicted from its sequence alone, underscoring the importance of using chemical modification data for RNA structure modeling. We find that Fox2 binding sites are typically located near internal loops in sno-lncRNA2, suggesting a structural context for the interaction with this splicing modulator. We are currently expanding this approach towards the analysis of other lncRNA, including additional members of the sno-lncRNA family.
Yin, Q.-F., Yang, L., Zhang, Y., Xiang, J.-F., Wu, Y.-W., Carmichael, G. G., & Chen, L.-L. (2012). Long noncoding RNAs with snoRNA ends. Molecular Cell, 48(2), 219–230. doi:10.1016/j.molcel.2012.07.033.
Talkish, J., May, G., Lin, Y., Woolford, J. L., & McManus, C. J. (2014). Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA (New York, NY). doi:10.1261/rna.042218.113