Presentation at COMBIO 2016 in the small ORF/peptide symposium. hew I talk about our work on ascorbate (vitamin C) and out identification and characterisation of a small uORF that regulate translation on ascorbic acid and the relevance of this to Iron deficiency anemia.

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Where are all the small peptides and what
might they do?
Roger P. Hellens, QUT, Brisbane, Australia
@rogerhellens

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Micronutrient deficiency – hidden hunger
http://www.fao.org

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DALYs and deaths attributable to
deficiencies of key vitamins and minerals
Steur et al. Nature Biotechnology 33, 25–29 (2015)

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Mechanism of Vitamin C-mediated Iron
uptake
Free Radical Biology and Medicine (2014) 75, 69–83

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The problem of iron deficiency anemia can be viewed as one of inadequate iron bioavailability; with that in mind, it
has been suggested that the problem may be better thought of as one of vitamin C inadequacy. Combs J, Gerald F.
The Vitamins. 4 ed. Burlington: Elsevier Science; 2012
Ann N Y Acad Sci. 1980; 355:32-44.
Vitamin C and Iron Deficiency

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Fresh Fruit as a Cure for Scurvy
The Diffusion Of Innovation, Everett M. Rogers
1617 The Surgeon's Mate by John Woodall (p 160-176)
We have in our owne country here many excellent remedies generally knowne, as namely, Scurvy-grasse, Horse-Reddish
roots, Nasturtia Aquatica, Wormwood, Sorrell, and many other good meanes... to the cure of those which live at
home...they also helpe some Sea-men returned from farre who by the only natural disposition of the fresh aire and
amendment of diet, nature herselfe in effect doth the Cure without other helps." At sea”, he states that experience shows
that "the Lemmons, Limes, Tamarinds, Oranges, and other choice of good helps in the Indies... do farre exceed
any that can be carried tither from England
1747 James Lind, HMS Sailsbury. (130 years later)1
• cider,
• elixir of vitriol (sulphuric acid),
• vinegar,
• sea water,
• 2 oranges and
• 1 lemons, and a
• purgative mixture of spicy paste and barley water
1795 Scurvy eliminated in the British navy (48 years later)
1856 British Board of Trade forms a policy of using lemons and limes to
eliminate scurvy in the trading fleet. (61 years later, 239 in total)
1 - Lind J (1753). A Treatise of the Scurvy. London: A. Millar.

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Vitamin C, So Much More Than a
Cure For Scurvy
1920 – anti-scurvy factor called vitamin C
1933 - L-ascorbic acid discovered as anti-scorbutic factor (Haworth and
Szent-Györgyi)
1970 - Vitamin C and the Common Cold (Linus Pauling)
1980 - Vitamin C and Iron Bioavailability
2000 - Vitamin C and Cognition/Mood (Visser)
2015 – Vitamin C and some Cancer (Kaiser et al, Science 2015: 619)
2016 –Vitamin C and epigenetics

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VitC concentration in kiwifruit
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
Pistil 1WAA fruit 2WAA fruit 4WAA fruit 6WAA fruit 12WAA fruit Mature fruit
Ascorbate(mg/100gFW)
Vitamin C in kiwifruit

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Laing et al., PNAS (2004 and 2007)
Biosynthesis of Ascorbate

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Transients Expression of Enzymes in Vitamin C
Biosynthesis
# genes
expressed
(control) PMM GMP GME GGP GPP
0 32 ± 2
1 32 ± 1
35 ± 1
32 ± 1
223 ± 31
49 ± 14
2 34 ± 1
38 ± 3
542 ± 57
177 ± 24
3 34 ± 2
505 ± 46
539 ± 62
4 574 ± 31
680 ± 34
5 515 ± 53

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Over-expression of GGP in plants
0
50
100
150
200
wt 10 3 3b 5 6 9 9a
Fruitascorbate(mg/100
gFW)
Transgenic lines
Strawberry
0
20
40
60
80
100
120
140
160
180
wt vector line 1 line 2 line 3 line 4 line 5
Fruitascorbate(mg/100g
FW)
Tomato
0
0.5
1
1.5
2
2.5
TuberAscorbate(mg/gDW)
Potato
Bullet et al. Plant Biotechnology Journal (2012) 10, 390–397

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0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.05
LUC/REN
0
0.05
0.1
0.15
0.2
0.25
LUC/REN
0
0.5
1
1.5
2
2.5
3
LUC/REN
0
0.1
0.2
0.3
0.4
0.5
0.6
LUC/REN
GGP-LUC TT8-LUC GGP-TT8-LUC TT8-GGP-LUC
+ - + - + - + -VitC
Promoter
Transcriptional or Post-Transcriptional Regulation?
GGP-promoter GGP-UTR LUC
TT8-promoter TT8-UTR LUC
LUC
LUC
TT8-UTR
TT8-promoter
GGP-promoter
GGP-UTR
LUC:REN

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5’ UTR Alignment of GGP

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RNA fold – 5’UTR region of kiwifruit GGP

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G
G A
A T G
T C A
C T
C
Non-ATG Start Codons

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Laing et al. Plant Cell (2015) 27:772-786
A uORF with non-ATG Start (ACG [Thr]) is Highly
Conserved in 5’UTR of GGP
dicotyledonous
algae to angiosperms

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sORFs – Where are they in the annotation?
Hellens et.al TIPS (2015) 21:317-328

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Short open reading frames (sORF)
and small peptides

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Predicted small open reading frames

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Actual small open reading frames

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sORF - How do you spot the real ones?
• Gene families of small peptides
• Sequence homology between difference
species
• sORFfinder – Ka/Ks
• ORF vs. CDS – regulatory role
25-residue peptide (Poneratoxin) from Bullet
Ant Paraponera clavata

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sORFs could be everywhere
Hellens et.al TIPS (2015) 21:317-328

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Non-ATG start codons may be common
Ingolia NT, et al. Cell 2011, 147:789–802.

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0
50
100
150
A
Numberofmatchingsequences
B
0 500 1000 1500 2000
0
20
40
Distance along GGP in base pairs
0
Numberofmatching
B
0 500 1000 1500 2000
0
20
40
Distance along GGP in base pairs
RNAseqRibosomeprofiling
Using data from Plant Cell. 2013 25:3699-710
Ribosome Profiling of GGP

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Runway traffic jam. Sunday after the F1 race was the busiest time for departures of
private aircraft. At one point, 24 planes were waiting for take off (AUSTIN -
Bergstrom International Airport).

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A Model for Vitamin C Regulation

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Observed vs. expected peptide size distribution
0
0.5
1
1.5
2
2.5
3
3.5
4
0:24 25-50 50:74 75:99 100:124 125:149 150:174 175:199
Millions
all ATG
all ATG +
ncATG
Annotated

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A role of non-ATG

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Accumulation GGP transcript

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So What - Improve Genome Annotation
ENCODE - Nature 489, 52–55 (2012) . doi:10.1038/489052a

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So What – biofortification in commodity crops
Plant source Amount (mg
/ 100g)
Camu Camu 2800
Acerola 1677
Seabuckthorn 695
Mica Muro 500
Indian gooseberry 445
Rose hip 426
Baobab 400
Chili pepper (green) 244
Red pepper 190
Chili pepper (red) 144
Kiwifruit 90
Broccoli 90
Brussels sprouts 80
Kale 41
Cauliflower 40
Garlic 31
Spinach 30
Cabbage raw green 30
Potato 20
Cassava 21
Tomato 10
Banana 10
Carrot 9
Apple 8
Onion 7
Asparagus 6
Beetroot 5
Lettuce 4
Cucumber 3
Eggplant 2
Sweet potato 2
Rice 0

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Summary
• Lots of unannotated small ORFs
• Not all peptides start with an ATG
•The GGP uORF is responsible for the
translational regulation of GGP

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Acknowledgements
Willaim A. Laing
Sean Bulley
Roy Story
Marcela Martinez-Sanchez
Richard C. Macknight
Matthew A. W. Chisnall
Chris M. Brown
Sally Roden
Meredith Crisp