Wrinkles, gray hair, … Cap2 methylation on mRNA? It turns out that these are all signs of aging. Two elegant new methods to show that Cap1 gets slowly modified to Cap2 as mRNAs age. And just like boomers, they want to live the quiet life, calming down the innate immune system to prevent autoimmunity.
Although we’ve known about Cap2 methylation for about 50 “golden” years, no one has fully understood what it does. It seemed important, though, since mice die young when the gene required for the mod is deleted, and it affects lung cancer cell growth.
But cranky Cap2 wasn’t about to give up its secrets easily, and current methods to investigate it aren’t very easy. Enter the Jaffrey lab (Cornell University), who had a fresh take and developed CapTag-seq. Here’s how it works:
- Decap mRNAs and ligate ends to an adapter of Nm so it’s resistant to RNase T2
- Treat with RNase T2, which cuts up the mRNA, except after Nm, leaving behind “cap tags”
- Convert these into cDNA libraries and count ‘em
The team found that Cap2 amounts were different in various organisms and cell types, and Cap2 methylation didn’t seem to be at specific sequences.
In CapTag-seq, RNase T2 destroyed all of the mRNAs aside from the caps, so the duo didn’t know the identities of the mRNAs. So of course, they birthed yet another brand-spanking-new method, CLAM-Cap-seq. Here, after decapping:
- Reverse transcribe decapped mRNA to make cDNA-mRNA
- Ligate these together at the 5’end of the mRNA with CircLigase
- Treat with RNase T2, which chops away the RNA, except the cap tag, which is stuck to the cDNA
- Ligate an adapter to the cap tag and generate a sequencing library
The researchers showed that Cap2 methylation is mostly on stable transcripts, but it doesn’t make them stable. This method and a variation that included qPCR showed that mRNAs get Cap2 mostly because they are just hanging around getting old, and Cap2 methylation keeps increasing as mRNAs age.
So, what do these aged Cap2-methylated mRNAs do? Well, in mice without Cap2, interferon-stimulated genes were upregulated. So, all that Cap1 that wasn’t turning into Cap2 triggered the innate immune response. And the team showed that Cap2 dampens the effects of accumulating Cap1 (Cap2 turns down Cap1’s cranked-up tunes). Ultimately, Cap2 is marking self mRNAs so that the innate immune system doesn’t go rogue, wreaking havoc and spurring an autoimmune reaction. Senior author Samie Jaffery shares, “We think Cap2 methylation occurs slowly, rather than quickly, in order to reduce the chance it will end up cloaking fast-replicating viral RNAs.”
Visit your poor ol’ Cap2-methylated mRNAs (and while you’re at it, get off their lawn!) at Nature, February 2023.