L6: m6A methylation - writers, detection methods Flashcards
What is the epitranscriptome?
RNA modifications that do not alter sequence
>170 highly diverse RNA mods
Function is context dependent and modification-specific
Eg. snRNA (splicing), t/rRNA (translation)
Examples of diseases linked to RNA modifications?
Neurological disorders
Mitochondrial diseases
Cancer (many show dysregulation of m6A modification)
What are various mechanisms of RNA modification?
Many different mRNA transcripts modified (only a few modifications per transcript)
snoRNA mediated (site specific rRNA modification, many copies rRNA within cell)
List different techniques to measure RNA modifications?
- 2D thin layer chromatography (TLC)
- Detection by mass spec (liquid chromatography then MS)
- Detection by mutations in transcripts
- Primer extension assay (RNA modification causes roadblock and termination earlier)
How does 2D TLC detect RNA modifications?
RNA is digested with nuclease into small pieces and labelled to allow detection after separation
Single nucleotides can be separated with different solvents
Based on polarity or charge (modified nucleotides often have distinct properties so migrate differently than unmodified)
Cellulose TLC plate (stationary phase)
Two different solvents (run TLC in different directions)
How do mutations in transcript allow detection?
Adenosine can be deaminated to inosine
Inosine pairs with cytosine, thus is read as guanosine
If A modified, cannot be deaminated so still read as A
Where are RNA modifications?
Modifications abundant in rRNAs and tRNAs
Locations of tRNA and rRNA modifications?
tRNA
Can change properties and interactions
- Amino acid attachment site
- D and T loop
- Anticodon loop
- Anticodon (wobble bp changes which nucleotides tRNA pairs with)
rRNA:
>200 modifications of ribosome at functional regions
- PTC
- Inter subunit bridge
- Decoding centre
How is N6-methyladenosine (m6A) on mRNAs sequenced?
Isolate mRNAs with polA purification and fragmentation
IP with antibody coupled beads
Wash and elute
RNA-seq on IP and input mRNAs
Controls are essential
- Negative controls input
- Ideally transcripts with no m6A (ime4/)
Explain how m6A detection works in eTAM-Seq
TadA enzyme modifies adenosine to insoine by deamination reaction
m6A cannot be modified by TadA
m6A reamins A whilst I will be detected as G
Give an overview of m6A regulation
Adenosine modified into m6A by writers
Modification removed by erasers
Readers involved in functions of these modifications
Eg. pre-mRNA splicing, RNA exportation, decay, stabilisation, mRNA translation
What diverse roles foes m6A play in mRNA metabolism?
mRNA decay
Translation initiation
Alternative splicing
Nuclear processing and export
What physiological processes is m6A involved in?
Cell differentiation
Gametogenesis
Stress responses
Carcinogenesis
How are m6A writer multi-subunit complexes conserved?
human + Arabidopsis/drosophila + S. cerevisiae
- METTL3/Ime4 catalytic subunit
- METTL14 stabilises METTL3
- RBM15/Nito RNA binding
- WTAP scaffold
- VIRMA stabilises complex
- HAKAI E3 liase
- ZC3H13 important for activity and localisation
How can m6A modifications be erased?
Similar mechanisms to DNA and histone demethylases
In: 2-oxoglutarate (2OG), Fe II
Out: Succinate, CO2
Byproduct: formaldehyd
Note: 2OG = alphaKG in DNA demethylation
What is FTO and how is it involved?
Fat mass and obesity associated enzyme
Key eraser in epitranscriptomic regulation
Regulates body weight
- FTO mutations increased risk of obesity
- FTO over-expressed in cancer
Chemical molecules used to inhibit FTO for potential therapeutics
Role of m6A writer complex in yeast meiosis?
m6A mRNA most abundant during early meiosis in yeast (detected in 1000 mRNAs)
Ime1 = m6A methyltransferase (writer) responsible for regulating meiotic progression
How was CoIP-MS of Mum2 used to identify new interactions?
- Grow cells
- Snap freeze and break cells
- Cell lysis and clarification of lysate
- Immunoprecipitation against Mum2
- TEV protease elution
- Protein identification through MS (what are they interacting with?)
What did CoIP-MS with Mum2 show?
Compare IP with Mum2 to control IP
Identify several proteins not known to be part of the complex
- Ygl036w uncharacterised at the time
- Npl3, Pab1 RNA binding proteins
- Dyn2 Dynein light chain protein
Explain results from repeat CoIP-MS experiment involving RNase?
Removes all RNA dependent interactions
Pab1 and Npl3 lost
Slz1 and Dyn2 remain
Ime4, Ygl036w, Kar4 remain
What conclusions were made after revisiting the budding yeast m6A writer complex?
Ygl036w (budding yeast) orthologous to VIRMA (mammalian)
Renamed to Vir1
Slz1 shows similarity to ZC3H13
Conclusion: budding yeast m6A writer conserved
What is the function of subunits?
Expect Dyn2, all are essential for m6A
Ime4 is catalytic subunit
What experiment tested the role of subunits stabilising each other?
Delete one subunit and test expression of others
Ime4, Mum2, Vir1 affect other components protein levels
Yeast m6A writer contains stabilising core of Ime4, Mum2, Vir1 (important role of subunits in complex stabilisation)
What is the function of m6A writer complex?
Facilitating yeast to undergo meiosis or spore formation
