Existential Epigenetics: Deciphering the Where, the Why, and the How of m6A and m6Am mRNA Methylation!

Can we break the speed of light? Is the universe infinite? What is the answer to life, the universe, and everything? At least two of these questions have fascinated physicists and philosophers for decades, but the questions weighing heavy on their minds of the existential epigeneticists among us relate to something a little different: the how, the where, and the why of mRNA methylation!

Researchers have generally focused on two specific types of mRNA methylation: the  N ⁶-methyladenosine (m⁶A) modification found within mRNA bodies, and the perhaps less appreciated N ⁶,2′- O-dimethyl adenosine (m⁶Am) modification encountered on the first transcribed nucleotide adjacent to the m⁷G cap. For m⁶Am specifically, two recent studies sought to describe the enzymes that control m⁶Am deposition (how!?), to distinguish m⁶Am from m⁶A and map the prevalence of m⁶Am throughout the transcriptome (where!?), and to evaluate the consequences of the m⁶Am modification (why!?).

Furthermore, a “bonus” paper also revealed some fascinating insight into the role of mRNA methylation, suggesting that liquid-liquid phase separation into distinct cytoplasmic compartments may control the translation of highly modified mRNAs!

Finding and Losing the PCIF Methyltransferase Enables Epigenetic Explorers to Map m⁶Am!

Our first unquestionably excellent study comes from an investigational team led by Samie R. Jaffrey (Cornell University, New York, USA) and Eric Lieberman Greer (Boston Children’s Hospital, USA) who employed a bioinformatic approach to identify a candidate m⁶Am methyltransferases and then construct a high-confidence transcriptome-wide map of both m⁶Am and m⁶A.

Boulias, Toczydlowska-Socha, Hawley, and colleagues set off without questioning their epigenetic abilities and soon uncovered some clues to the how, the where, and the why of mRNA methylation:

• Bioinformatic analyses of orphan adenosine methyltransferases, cap-binding assays, and in vitro methyltransferase assays combined with ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry identified Phosphorylated CTD Interacting Factor 1 (PCIF1) as an m⁷G cap-binding-dependent m⁶Am methyltransferase
  • The evolution of PCIF1 at the same time as the mRNA 5´ cap emerged provides further evidence for PCIF1 as an m⁶Am methyltransferase
• PCIF1 knockout in normal and cancer cells abolished m⁶Am levels without affecting m⁶A
  • m⁶A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) assays in PCIF1 knockout cells permits the distinction of m⁶Am from m⁶A modifications and the creation of site-specific high-confidence transcriptome-wide maps
  • These new maps highlight errors in previous mRNA methylation annotations that they now know reflect the existence of mRNA isoforms that differ by transcription start sites; some isoforms contain transcriptional start sites that map to internal sites previously attributed as the m⁶A modification
• Analysis of PCIF1 knockout cells demonstrates that m⁶Am increases the stability of some mRNAs, but has minimal effects on translation under basal conditions

The authors hope that the identification and characterization of PCIF1 as an m⁷G cap-binding m⁶Am methyltransferase coupled with precise m⁶Am annotations generated by PCIF1 knockouts will answer the existential question of why m⁶Am exists a wide range of cell types.

For more on how this team of epigenetic explorers created mammalian cell maps of m⁶Am and sought to discover its consequences, see Molecular Cell, July 2019.

PCIF1 – The “One and Only” m⁶Am Methyltransferase

Our second highly inquisitive study comes not from the 80’s one-hit-wonder Chesney Hawkes, but instead a team of researchers led by Yang Shi (Boston Children’s Hospital, USA), who also sought to discover more about the m⁶Am mRNA modification and now report PCAF1 as the “one and only” m⁶Am methyltransferase.

Sendinc and colleagues set out on their exploratory path to prove they are “not the same as all the rest” and confirmed the how and the where of m⁶Am in various cancer cell types and, in doing so, potentially discovered the why by demonstrating a putative mechanism of gene expression regulation involving PCIF1:

• The authors confirmed the evolutionary conservation of m⁶Am in mRNA of vertebrate organisms, which they tested through sensitive mass spectrometry-mediated detection/quantification
  • However, m⁶Am is not present in fission yeast, nematodes, or Drosophila
• Gene knockouts, thin layer chromatography of mRNAs, in vitro methylation assays, and rescue experiments confirmed that PCIF1 mediates m⁶Am in an m⁷G cap-dependent manner
  • Deletion of PCIF1 results in the complete loss of m⁶Am from mRNA, and therefore PCIF represents the “one and only” m⁶Am methyltransferase in human cells
  • PCIF1 knockout leads to m⁶Am loss but fails to affect m⁶A levels or distribution
• The development of an exonuclease-assisted high-throughput sequencing methodology (m⁶Am-Exo-Seq) permitted the generation of m⁶Am distribution maps
  • The rationale for this technique is that the enrichment of mRNA 5’ ends would enrich for m⁶Am and deplete the internal m⁶A loci
  • Analysis of these maps reveals the independence of m⁶Am and m⁶A, suggesting a functional distinction between these mRNA modifications
• Using precision nuclear run-on sequencing and RNA-sequencing combined with m⁶Am-Exo-Seq analyses in wild-type and PCIF1 null cells, they established that the presence of m⁶Am does not affect mRNA stability
  • Instead, reporter assays using modified mRNA transcripts and quantitative proteomics analyses suggest that m⁶Am negatively affects cap-dependent translation of methylated mRNAs

Overall, this team of mRNA methylation maestros not only confirmed the previous studies findings but also show that PCIF1 is the “one and only” m⁶Am methyltransferase in mammalian cells.

For more on this smash-hit epigenetic study from a lab of roving researchers (surely they will not be one-hit-wonders!), see Molecular Cell, July 2019.

Methylation Takes mRNA to the Next Phase

The productive team of epigenetic trekkers led by Samie R. Jaffrey (Cornell University, New York, USA) was also behind our “bonus” mRNA methylation study, which sought to understand how methylation affects mRNA fates and why said effects seem to differ under distinct cellular contexts.

Fascinatingly, Ries and colleagues discovered that methylation allows for the compartmentalization of mRNA into specific regions through liquid-liquid phase separation (LLPS) thanks to binding by a family of related proteins:

• Analysis of purified full-length recombinant YTHDF1, 2, and 3 and photobleaching experiments revealed that these cytosolic m⁶A-binding proteins undergo LLPS
• Elevated levels of m⁶A methylation on an engineered RNA induces the binding of multiple YTHDF proteins and promotes LLPS via interactions between the low-complexity domains of the YTHDF proteins and enrichment in phase-separated cytoplasmic compartments
  • However, mRNAs lacking m⁶A modification or those with only a single m⁶A modification do not permit LLPS, and such mRNAs display low enrichment in phase-separated cytoplasmic compartments
• Stress induction causes m⁶A-modified mRNA-YTHDF complexes to localize to phase-separated cytoplasmic compartments
  • Interestingly, m⁶A-modified mRNA-YTHDF2 complexes move from one type of compartment (P-body) in unstressed cells into another (stress granules) after stress induction, suggesting that YTHDF2 allows partitioning into different structures
  • YTHDF2 binding to m⁶A-modified mRNA is strictly required to efficiently partition complexes into phase-separated cytoplasmic compartments
• YTHDF-mediated LLPS-mediated partitioning of m⁶A-modified mRNAs into cytoplasmic compartments does not induce mRNA degradation
  • Instead, highly m⁶A-modified mRNAs remain preferentially repressed, perhaps due to their compartmentalized state

This exciting new study suggests that variations in m⁶A levels within different cell, disease, differentiation or signaling contexts can influence the final transcriptomic output by inducing LLPS of those mRNAs with higher levels of m⁶A and thereby altering their translation. Furthermore, the authors note that the regulation of the YTHDF proteins themselves may permit an additional level of control.

For more on how m⁶A takes mRNA to the next phase, head over to Nature, July 2019.

What is the Next Question for mRNA Methylation?

The identification of PCIF1 as an m⁶Am methyltransferase has fostered an enhanced appreciation of the roles of site-specific mRNA methylation while the newly described phase separation of highly methylated mRNAs provides us with a possible consequence that may affect the translation of genes under specific conditions.

The maps have been drawn up, and the scouting parties from several labs have sent back encouraging reports that we will soon discover unexplored epigenetic realms with many more untold stories regarding mRNA methylation. Stay tuned for all the new and exciting breakthroughs!