Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions
Ruijter, Anna Elisabeth Maria
Nature Publishing Group
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Stojic, L., Niemczyk, M., Orjalo, A., Ito, Y., Ruijter, A. E. M., Uribe-Lewis, S., Joseph, N., et al. (2016). Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions. Nature Communications, 7 (10406)https://doi.org/10.1038/ncomms10406
Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.
long noncoding RNA, transcriptional interference, siRNA, gene regulation
The authors acknowledge all the members of Murrell, Rinn, Odom and Gergely laboratory as well as Massimiliano di Pietro, Klaas Mulder, Anna Git, Jason Carroll in Cambridge and Laurence Hurst (University of Bath) for reading and providing helpful comments on the manuscript. We also thank the Genomics, Microscopy and Bioinformatics core facilities at the Cambridge Institute for support, Christina Ernst for thumbnail image design, Ezgi Hacisuleyman for the design of the negative control vector, Cole Trapnell and David Hendrickson for providing us with lincExpress vector, Arjun Raj with the RNA FISH and Alaisdair Russell with the lentiviral work. This research was supported by The University of Cambridge, Cancer Research UK and Hutchison Whampoa Limited. The authors have no conflicting financial interests.
Cancer Research UK (C14303/A17197)
European Research Council (615584)
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External DOI: https://doi.org/10.1038/ncomms10406
This record's URL: https://www.repository.cam.ac.uk/handle/1810/252719
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/