19.01.09 Nonsense mediated mRNA delay (NMD)

Question 1

What are mRNA surveillance pathways?

Answer 1

Quality control processes that exist to protect eukaryotyic cells by reducing the production of potentially harmful proteins from aberrant mRNA transcripts

Question 2

Name 3 mRNA surveillance pathways

Answer 2

1) Nonsense Mediated mRNA decay pathway (NMD) - targeting transcripts containing premature termination codons (PTCs) 2) Nonstop Mediated mRNA decay pathways (NSD) -  mRNA transcripts which lack a stop codon 3) No-go Mediated mRNA decay pathway (NGD) -  mRNA transcripts on which ribosomes have stalled (e.g. due to secondary structures)

Question 3

Nonstop Mediated mRNA decay pathway (NSD)

Answer 3

• Detection and decay of mRNA transcripts which lack a stop codon (this causes significant issues for cell)
• Ribosomes translating the mRNA eventually translate into the 3’poly-A tail region of transcripts and stalls and can therefore not eject the mRNA.
• Ribosomes would not be available to translate other mRNA molecules into proteins.
• NMD solves this issue by freeing the stalled ribosomes, and marking the mRNA for degradation by nucleases

Question 4

No-go Mediated mRNA decay pathway (NGD)

Answer 4

• Most recently discovered mechanism so not well understood
• Targets mRNA transcripts on which ribosomes have stalled during translation

Question 5

Nonsense Mediated mRNA decay pathway (NMD)

Answer 5

• Targets transcripts containing PTCs (caused by germline variants, somatic variants, errors in transcription or post-transcriptional mRNA processing
• Mechanism protects again deleterious dominant-negative and gain of function effects
• PTC must be recognised as different from normal stop codon
• NMD NOT predicted to occur id PTC occurs in 3’ most exon or within the 3’-most 50 nucleotides of the penultimate exon

Question 6

What is the main rule of NMD?

Answer 6

NMD is predicted to occur if the PTC is upstream of the 3’ most 50 nucleotides of the penultimate exon.

Question 7

What is the Exon-junction complex (EJC) Model?

Answer 7

• Multiprotein complexes that assemble during splicing 20-24 nucleotides upstream from splice junction (marks exon-exon boundary)
• Exon-exon boudnary marks sites where introns were spliced out
• As ribosome moves along mRNA it displaces any EJC in its path. Once at the termination codon, release factors interact with any undisplaced EJCs which triggers decapping of transcript and degradation (so any with PTCs will have EJCs remaining which highlights the transcript for degradation by NMD)
• EJC normally stripped off mRNAs by the translating ribosome
• If a PTC is present the EJC remains bound after the first round of translation - The interaction between termination factors and the downstream EJC triggers NMD
• Elongating ribosome reaches a PTC and a complex is formed of SMG1, UPF1, eRF1, eRF3 (SURF complex)
• SURF then interacts with UPF2, UPF3b and an EJC downstream of the PTC to form the mRNA decay-inducing complex (DECID)
• UPF1 (ATP-dependent RNA helicase) is phosphorylated and activated by SMG1
• Phosphorylated UPF1 acts to recruit SMG5, SMG6 and SMG7, and general mRNA degradation factors that lead to mRNA degradation

Question 8

What are the exceptions of the NMD rule?

Answer 8

1) T cell receptor gene rearrangements. Transcripts degraded by NMD despite having a PTC located within the last 50 nucleotides of the penultimate exon.
2) a long 3’ UTR can also target a transcript for NMD; e.g SMG-5 transcripts are regulated by NMD by virtue of its long 3’UTR - 3’UTR involvement could act as fail-safe mechanism to eliminate mutant transcripts which would otherwise escape degradation.
3) PRKAR1A mutations causing Carney complex (multiple endocrine neoplasia syndrome) in the last coding exon are predicted to escape NMD, however instead undergo NMD (Patronas et al. 2012).
4) β-thalassaemia- transcripts containing nonsense mutations in exon 1 β-globin escape NMD

Question 9

Disease example - β-thalassaemia

Answer 9

• Group of inherited anaemias caused by PTCs in 1st or 2nd exon of three-exon gene: β-globin
• HETs are asymptomatic (low or absent mutant mRNA)
• However, subgroup of HETs have more severe phenotype (thalassemia intermedia) - due to truncated non-functional proteins that are toxic
• Also - β-globin mRNAs with nonsense pathogenic variants in the first exon do not cause NMD if they are located from codons 2-23 (whilst those found from codon 26 down are degraded as expected)

Question 10

Disease example - DMD and BMD

Answer 10

• DMD - more severe phenotype. Variants disrupt reading frame and mRNA degraded by NMD - BMD - Less severe. Variants maintain reading frame, so protein still produced (but abnormal and less of it)

Question 11

Disease example - Marfan syndrome

Answer 11

• AD connective tissue disorder - Variants in FBN1 gene - Dominant negative disease (between mutant and wildtype monomers)- so nonsense variants cause a mild phenotype

Question 12

Name 3 potential treatments for PTCs?

Answer 12

1) Read through drugs - read through PTC to generate full length protein - here aminoacyl tRNA that has 2 out of 3 complementary nucleotides anneals to the termination codon in error and is incorporated into the polypeptide, generating the full length peptide.
- These have been attempted to acheive sufficicent protein levels, which in vivo show good effects but clinical trials have been mixed
2) Splice-switching oligonucleotides - restore normal splicing and induce exon skipping - can be used to correct reading frame in DMD
3) NMD inhibitors - prevnt UPF1 phosphorylation and NMD
- These are helpful, as NMD normally removes all mutatn mRNA and this is needed for other therapies to work (i.e. read through drugs)
- However therapeutic use is not yet proven