Life can be hard out there for an X chromosome, particularly when faced with a well-known molecular form of sexual discrimination. The key to this repressive environment and resulting existential crisis is X-inactive specific transcript (XIST), a long non-coding (lnc)RNA known to undergo modifications such as cytosine methylation.
However, RNA methylation is a rapidly evolving field and just like with its DNA counterpart, there is more than one type of methylation. In case your memory needs a refresher, N6-methyladenosine (m6A) is a dynamic RNA modification with great regulatory potential, and while there are a number of studies examining m6A in mRNA, its role in lncRNA has remained a functional mystery.
In order to tackle this mystery, the labs of Samie Jaffrey at Cornell University and Mitchell Guttman at the California Institute of Technology have teamed up to bring new insight into the functional mechanisms behind XIST mediated gene silencing. Previously, while screening for proteins that bind XIST, the team came across RNA-binding motif protein 15 (RBM15) and they now sought to investigate RBM15 along with its paralogue RBM15B. Here’s what went down:
RBM15 and RBM15B Bind to XIST
- They interrogated human embryonic kidney 293T (HEK293T) cells with individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to uncover that both RBM15 and RBM15B bind to XIST in a similar manner
- An engineered mouse embryonic stem cell (mES) line allowed for quantitative expression analysis of two X-chromosome genes by single molecule RNA FISH, revealing that:
- siRNA knockdown of RBM15 or RBM15B alone doesn’t have a significant effect on X-inactivation
- However, the double knockdown prevents XIST mediated gene silencing, which brings the redundant functions of RBM15 and RBM15B to light
m6A and its Machinery are Critical for XIST Mediated Silencing
- Immunoprecipitation experiments revealed that RBM15 and RBM15B co-precipitate with methyltransferase like 3 (METTL3), an m6A methyltransferase, thus helping METTL3 find its target sequence.
- A re-examination of their past data from m6A iCLIP (miCLIP) in HEK293 cells revealed 78 m6A sites in XIST, which is more than any other RNA
- By using m6A-RNA immunoprecipitation in their mES line, they found that siRNA knockdown of METTL3 decreases m6A methylation in XIST and interferes with its gene silencing function
- They then investigated the recognition of m6A on XIST using iCLIP, revealing that YTHDC1 binds XIST at m6A sites and YTHDC1 knockdown impairs XIST mediated silencing
- Finally, they engineered a clever system to tether YTHDC1 to XIST and found that when the two are together, X chromosome silencing occurs even in the absence of the regularly needed m6A machinery
The Molecular Mechanism of XIST Mediated Silencing via m6A
Taken together, the team’s results show that RBM15 and RBM15B bind to specific sequences on XIST and promote m6A formation via a complex with METTL3. The presence of m6A on XIST then allows for binding of YTHDC1, which enables repression of the X chromosome.
Get the full functional picture of the role of m6A on XIST in Nature, September 2016