L7: m6A methylation - readers and regulation

Question 1

General overview for readers of m6A methylation?

Answer 1

Dynamic m6A marks are read by reader proteins that transduce signal to regulate mRNA metabolism and translation

Question 2

Sentence including writers and erasers for m6A methylation?

Answer 2

m6A methylation of transcriptome defined by activity of methyltransferases (writers) and demethylases (erasers) in nucleus

Question 3

What did key studies in 2011/12 show about m6A?

Answer 3

Reversible so can be regulated
Domonissini et al showed as widespread mechanism of RNA regulation that responds to cell regulatory pathways

Question 4

What is important to remember about research into m6A methylation?

Answer 4

Field advancing fast and many important features still debated (contradictions may be context dependent)

Question 5

What is Me-RIP-seq?

Answer 5

m6A RNA immunoprecipitation sequencing

Uses antibody that specifically recognises m6A modified RNA
Isolates m6A modified RNA and identifies where m6A occurs across transcriptome

Question 6

What does Mr-RIP seq reveal about m6A sites

Answer 6

Provides low resolution (~100 nt) map of m6A methylation in transcriptome
Does not pinpoint exact bases
Represent the averaging of the mRNA population

Question 7

What were the key findings from the first MeRIP-Seq dataset?

Answer 7

~13,000 m6A sites identified in over 6000 genes
Shows m6A methylation is widespread and likely functionally important

Question 8

Where are m6A modifications enriched in mRNAs?

Answer 8

Distribution of sites in transcriptome not random
- Skewed towards stop codon region and within 3’ UTR

Question 9

What regulatory roles were suggested by MeRIP-Seq data?

Answer 9

Some m6A sites found to be regulated by cell signalling pathways
Hypothesised m6A can influence RNA splicing

Question 10

What did early biochemical assays reveal about m6A reader proteins?

Answer 10

Identified set of m6A binding proteins that contain YTH motif

Question 11

What is the YTH motif?

Answer 11

Conserved structural motif that directly recognises m6A

Question 12

How were m6A binding proteins discovered?

Answer 12

mRNA pull downs and MS
- Identified number of proteins associated to m6A methylated RNA element

Question 13

What 5 YTH-containing proteins were found in humans?

Answer 13

1,2,3
- 3 paralogues
- Very similar domain organisation
- Partially overlapped set of targets but perform different functions
- Contain long low-sequence complexity regions

4
- Nuclear YTH protein
- Different domain organisation
- Also comprises long low-sequence complexity regions

5
- Highly structured YTH protein
- Thought not to recognise m6A

Question 14

How are the 3 (YTH)DF1 proteins conserved in vertebrates and invertebrates?

Answer 14

Similar to other important RNA regulators, 3 YTH paralogues present and conserved in vertebrates

Single member of the family present in invertebrates

Question 15

How do human YTH proteins recognise the m6A group?

Answer 15

Using an aromatic cage
- Methyl group recognised via hydrophobic interactions within aromatic cage

Question 16

What provides specificity for recognising only m6A?

Answer 16

• Interactions of protein with other base moieties guarantee selectivity of A methylated in position (N) 6
• H-bonding of sugar 2’O moiety discriminates against m6A

Note: some further specificity present for bases flanking m6A but recognition not essential

Question 17

What are the regulatory functions of YTH proteins?

Answer 17

YTH proteins regulate different steps of mRNA metabolism and translation

Same YTH protein can act upon different mechanisms by interacting with different RNA regulatory factors
- Regulatory function probably context dependent

Question 18

How can RNA binding proteins (RBPs) act as m6A readers?

Answer 18

Some multi-functional RBPs, which recognise RNA, can act as m6A even without YTH motif

Question 19

How does presence m6A influence RNA-protein interactions?

Answer 19

m6A could change or destabilise RNA structure
- Can activate or block interactions with specific protein regulators

Question 20

How does direct recognition of m6A affect RNA?

Answer 20

Trans-acting proteins could directly recognise m6A
- Regulate different aspects of processing and degradation

Question 21

What is hnRNPC and its function?

Answer 21

RNA binding protein that recognises polyU sequences in pre-mRNA
Regulates alternative splicing (known splicing regulator)

Question 22

How was influence of hnRNPC investigated?

Answer 22

RNA pull down assay using cell extract
- Found that hnRNPC interaction strength is m6A dependent

Question 23

What is the mechanism of hnRNPC?

Answer 23

Once bound, may alter the splicing process, resulting in various protein isoforms

Question 24

Which experiments revealed the hnRNPC recruitment and binding mechanism?

Answer 24

Combined PAR-CLIP meRIP assay
RNA protection assays

Question 25

What evidence supports structure-mediated mechanism of m6A regulation?

Answer 25

Sequence analysis - From combined PAR-CLIP ad MeRIP-seq - Revealed enrichment and overlap of both m6A consensus sequence and hnRNPC binding site sequences (polyU) Suggests m6A methylation enhances hnRNPC recruitment

Question 26

What did RNA protection assays show on hnRNPC recruitment?

Answer 26

Methylation exposes 2 additional cleavage sites Indicates local RNA helix destabilisation and opening

Question 27

What model was proposed for how m6A methylation mediates hnRNPC recruitment?

Answer 27

m6A 'switch' model proposes: - m6A disrupts secondary structure, exposing polyU sequences - Enables hnRNPC to bind and regulate alternative splicing

Question 28

What are IGF2BP proteins and how do they relate to m6A?

Answer 28

IMP1-3 RNA binding proteins - Recognise and bind m6A modified RNAs - Both in vitro and vivo

Question 29

What is the domain structure of IGF2BP (IMP) proteins?

Answer 29

Contain 6 putative RNA binding domains - 4 KH domains - 2 RRM domains

Question 30

KH4 shows high specificity for what motif?

Answer 30

GGAC Recognised with high specificity in vitro

Question 31

What experiment supports IGF2BPs selective binding to m6A?

Answer 31

RNA affinity chromatography - Show IGF2BP preferentially bind m6A modified RNA - Binding lost when m6A marks removed

Question 32

Explain the methyltransferase knockdown experiment?

Answer 32

1. Used shRNA-mediated silencing of m6A specific methyltrasnferase (eg. METTL3) 2. Decreased m6A levels 3. Impaired binding to high confidence target mRNAs by CLIP (crosslinking and immunoprecipitation)

Question 33

What is the functional role of IGF2BP-m6A interaction?

Answer 33

IGF2BP regulate stability of target mRNAs in m6A-dependent manner - Without m6A, IGF2BP proteins bind less effectively to target mRNAs

Question 34

Experiment for which proteins are involved in m6A regulated IMP-mediated stabilisation of c-Myc?

Answer 34

Computational analysis - Compare m6A sites on transcriptome with known interaction sites of RNABP - Identified proteins interacting in proximity of methylated sequence - ELAV1/HuR interacts near IMP1

Question 35

What did coIP assays show about HuR and IMP1?

Answer 35

Indicated HuR is part of IMP1 complex

Question 36

What did immunofluoresence show about HuR and IMP1?

Answer 36

Found IMP1 and HuR colocalised in cytoplasmic granules

Question 37

How does IMP1 recognise m6A?

Answer 37

Using KH34 domain Through hydrophobic interactions in shallow cradle

Question 38

What was the set up of the BLI experiment for IMP1-m6A?

Answer 38

Immobilised IMP1 KH34 domain on chip Exposed to different IMP1 concentrations Can obtain information on kinetics and affinities

Question 39

What were the results from the BLI experiment on IMP1-m6A

Answer 39

m6A methylation increases IMP1 binding lifetime 8-fold and affinity 5-fold for ACTB RNA

Question 40

What is the difference between IMP1-m6A and YTH-m6A recognition?

Answer 40

IMP1 recognition is concentration dependent YTH domains discriminate effectively regardless of concentration

Question 41

How do mutations in IMP1 affect m6A binding?

Answer 41

Mutation in hydrophobic cradle - Inverts IMP1 specificity for m6A RNA

Question 42

What is the purpose of comparing mutant IMP1 to WT IMP1?

Answer 42

Mutant IMP1 can be used to assess m6A regulation through comparison with WT in iCLIP and functional assays

Question 43

What is m6A regulation dependent on?

Answer 43

Context dependent Varies across cell type and developmental stage