Premature termination codons (PTCs) cause a large proportion of inherited human genetic diseases. PTC-containing transcripts can be degraded by an mRNA surveillance pathway termed nonsense-mediated mRNA decay (NMD). However, the efficiency of NMD varies; it is inefficient when a PTC is located downstream of the last exon junction complex (EJC). We used matched exome and transcriptome data from 9,769 human tumors to systematically elucidate the rules of NMD targeting in human cells. An integrated model incorporating multiple rules beyond the canonical EJC model explains approximately three-fourths of the non-random variance in NMD efficiency across thousands of PTCs. We also show that dosage compensation may sometimes mask the effects of NMD. Applying the NMD model identifies signatures of both positive and negative selection on NMD-triggering mutations in human tumors and provides a classification for tumor-suppressor genes.
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Rules and Impact of NMD in Cancers
1. THE RULES AND IMPACT OF
NONSENSE-MEDIATED mRNA DECAY
IN HUMAN CANCERS
Rik Lindeboom, Fran Supek*, Ben Lehner*
EMBL/CRG Systems Biology Unit, Centre for Genomic Regulation,
Barcelona
published as: Lindeboom et al. Nature Genetics 48, 1112–1118 (2016)
2. Nonsense-mediated mRNA decay
(NMD) pathway
• reduces the production of harmful truncated
proteins, translated from nonsense variant-bearing
transcripts
• NMD is involved in post-transcriptional regulation
of gene expression (physiological role) in ~5%
genes
• nonsense variants cause many human genetic
diseases, making NMD an important modulator of
disease outcome
3. NMD ameliorates disease phenotypes (e.g. in beta-thalassemia)
Beta-globin: heterozygous nonsense mutations in
... exon III -> mutant + normal mRNA -> w.t + truncated protein -> thalassemia
... exon II -> no mutant mRNA -> only w.t. protein -> asymptomatic
Peixeiro et al (2011) Haematologica
4. NMD protects (in beta-thalassemia)
NMD aggravates phenotypes
(in Duchenne muscular dystrophy)
5. Known models of NMD
„Faux 3’UTR model” – yeast,
Drosophila, C. elegans
Wen & Brogna 2008
6. Known models of NMD
„Faux 3’UTR model” – yeast,
Drosophila, C. elegans
Wen & Brogna 2008
Exon-junction complex
model – the above
organisms, plus mammals
Peixeiro et al (2011) Haematologica
EJC
7. Known models of NMD
„Faux 3’UTR model” – yeast,
Drosophila, C. elegans
Wen & Brogna 2008
Exon-junction complex
model – the above
organisms, plus mammals
Known exception: individual examples of PTCs (premature termination codons) close to the
start codon, rendering the transcripts insensitive to NMD (regardless of downstream EJCs!).
Peixeiro et al (2011) Haematologica
Peixeiro et al (2011) Haematologica
8. These rules were found by previous work examining small sets (n≈1-10) of reporter gene
variants, in many individual experiments.
We set out to find: (a) how widely they apply to human genes? (b) are there other rules?
Cancer genomes an invaluable resource to systematically elucidate the rules of NMD.
figures from Lindeboom, Supek and Lehner. Nat Genet 2016
9. A downstream EJC and proximity to the start codon are
indeed widespread signals for NMD in human genes.
10. A downstream EJC and proximity to the start codon are
Indeed widespread signals for NMD in human genes.
Penultimate
exon can also be
protected from
NMD (if the last
exon is non-coding)
Evidence for the
translation
re-initiation
hypothesis
NMD evasion at 5’ end
Splice site in 3’ UTR
(individual examples
were known; we
show systematic
trend)
(novel)
figures from Lindeboom, Supek and Lehner. Nat Genet 2016
11. New influences on NMD efficiency that we discovered:
Exon length, mRNA decay rate and RNA-binding proteins
12. New influences on NMD efficiency that we discovered:
Exon length, mRNA decay rate and RNA-binding proteins
Peixeiro et al (2011) Haematologica
short dist.
to 3’ EJC
long distance
to 3’ EJC ->
The EJC model of NMD:
... plausibly could
make their interaction
more difficult
13. New influences on NMD efficiency that we discovered:
Exon length, mRNA decay rate and RNA-binding proteins
Fast turnover = less efficient NMD
(competing processes)
14. New influences on NMD efficiency that we discovered:
Exon length, mRNA decay rate and RNA-binding proteins
Aconitase 1 (ACO1 or IRP1) is a metabolic enzyme that is known to also
regulate gene expression via binding to UTRs of many genes.
The presence of the ACO1 motif in the 3′ UTR decreases NMD efficiency
(p=0.023, 16%–24% less efficient NMD), suggesting that the ACO1-
dependent mRNA turnover rate might modulate NMD efficiency.
- validates across 3 data sets -
one example:
+8 other motifs with similar support
15. New influences on NMD efficiency that we discovered:
Exon length, mRNA decay rate and RNA-binding proteins
ACO1 motifs are generally
associated to rapid mRNA turnover
(...thus less efficient NMD –
as observed generally)
Aconitase 1 (ACO1 or IRP1) is a metabolic enzyme that is known to also
regulate gene expression via binding to UTRs of many genes.
The presence of the ACO1 motif in the 3′ UTR decreases NMD efficiency
(p=0.023, 16%–24% less efficient NMD), suggesting that the ACO1-
dependent mRNA turnover rate might modulate NMD efficiency.
- validates across 3 data sets -
Also, irrespective of stop codons....
16. Estimating the contributions of various factors to
global NMD efficiency (using Random Forest regression)
Known:EJC
model
novel
rules
yettobe
elucidated
17. Estimating the contributions of various factors to
global NMD efficiency (using Random Forest regression)
(rough estimates of dosage compensation
in human cells by comparing somatic copy-
number alterations with mRNA levels)
Known:EJC
model
novel
rules
yettobe
elucidated
18. Signatures of negative and positive selection on somatic nonsense mutations.
effects of NMD provide a novel
classification of tumor suppressor genes
evidence that negative (purifying) selection
acts on nonsense somatic mutations
Lindeboom, Supek and Lehner. Nat Genet 2016
19. Signatures of negative and positive selection on somatic nonsense mutations.
9 novel tumor
suppressor genes (TSG)
proposed by Davoli
et al (2013) Cell
we think that the novel TSGs
are likely to act in a
haploinsufficient manner
Lindeboom, Supek and Lehner. Nat Genet 2016
20. Rik Lindeboom
funding from:
CRG Core, ERC, 4dCellFate,
Severo Ochoa, AXA,
AGAUR, Croatian Science Foundation
overview of Lindeboom, Supek and Lehner. Nat Genet 2016
Editor's Notes
The rules of NMD were inferred from nonsense somatic mutations (n = 2,840) in TCGA tumor genome sequences and then validated using somatic frameshift changes (n = 3,151) in TCGA and germline nonsense variants (n = 1,784) in Geuvadis. NMD efficiency was quantified via mRNA expression levels for TCGA (after control for somatic copy number changes) and via allele-specific expression for the Geuvadis cohort. WT, wild type.