Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome.
A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications.
Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Kavitha
K
Sarma
Pierre
P
Levasseur
Alexander
A
Aristarkhov
Jeannie T
JT
Lee
D000818:Animals; D002461:Cell Line, Transformed; D044127:Epigenesis, Genetic / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D063146:Polycomb-Group Proteins; D020871:RNA Stability / Q000502:physiology*; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014960:X Chromosome / Q000235:genetics / Q000378:metabolism*
D006657:Histones; D063146:Polycomb-Group Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; C077885:XIST non-coding RNA