When it comes to X inactivation there are a huge number of studies that eXist. Despite being one of the best-studied epigenetic phenomena, the molecular decision making process behind the initiation of X chromosome inactivation (XCI) had remained far from fully understood. The regulation of the long non-coding RNA Xist initiates XCI. Xist’s antisense partner and repressor Tsix is believed to regulate the choice of which X chromosome is inactivated in each cell. Tsix is initially biallelically expressed but then becomes silent on the inactive X, allowing Xist propagate and inactivate that copy. Therefore, understanding the regulation of the transition from biallelic to monoallelic Tsix expression is key to understanding XCI.
Characterizing the proteins interacting with RNAs expressed at low levels, like Tsix, is extremely challenging with most RNA-protein technologies. Existing RNA-protein interaction methods usually rely on UV crosslinking, which is not very efficient, and not sensitive enough for the low level of many RNAs, i.e. those involved in XCI. Thankfully, the lab of Jeannie Lee at Harvard Medical School developed a new technique for this purpose. They adapted BioID, a technique which uses a biotin ligase (BirA) to biotinylate proteins that are closely associated with a protein of interest. To adapt it for RNA, the authors fused BirA to a linker protein—phage PP7 coat protein (PP7cp)—which is then tethered to an RNA of interest by inserting PP7 RNA stem–loop motifs. They call the method BioRBP.
Here what they found:
- BioRBP identified proteins enriched for transcription- and chromatin-associated functions as Tsix-interacting proteins in mouse embryonic stem (ES) cells
- This includes DCP1A, an RNA decapping enzyme that is believed to regulate RNA stability
- In the nucleolus, DCP1A binds to Tsix and controls its stability
- Knock-down of DCP1A causes accumulation of X–X pairs (i.e. one X not being inactivated) and prevents the transition to monoallelic Tsix expression
- Using CRISPR-Cas9, they inserted specific tags in each Tsix allele in a mouse ES cell line lacking DCP1A expression and found that targeting each Tsix allele for degradation leads to upregulation of Xist in cis (on that chromosome)
- Finally, they investigated the role CTCF binding to decapped Tsix given its implication in their BioRBP experiment at the X-X pairing center:
- They found that Tsix RNA recruits CTCF to the pairing centre and the persistence of Tsix RNA prevents CTCF from engaging its DNA target
- They propose a model wherein DCP1A degrades Tsix RNA on the future inactive X to enable CTCF to bind chromatin, which then silences the Tsix allele in cis to enable Xist upregulation.
These new results further our understanding of role of RNA-protein interactions during XCI. Beyond these questions, BioRBP may also have uses in other contexts. Many developmental programs involve RNA expressed at very low levels in a small number of cells, BioRBP may provide a means to study these phenomena.
So take off your hat off to this complex mechanism over at Nature Cell Biology, August 2020.