roger hellens combio - oct 2016
TRANSCRIPT
CRICOS No. 00213J
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 ScurvyThe 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
Asco
rbat
e(m
g/10
0g F
W)
Vitamin C in kiwifruit
CRICOS No. 00213JLaing 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 ± 21 32 ± 1
35 ± 132 ± 1
223 ± 3149 ± 14
2 34 ± 138 ± 3
542 ± 57177 ± 24
3 34 ± 2505 ± 46
539 ± 624 574 ± 31
680 ± 345 515 ± 53
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Over-expression of GGP in plants
wt 10 3 3b 5 6 9 9a0
20406080
100120140
Strawberry
Transgenic lines
Frui
t asc
orba
te (m
g/10
0 g
FW)
wt vector line 1 line 2 line 3 line 4 line 50
2
4
6
8
10
12Tomato
Frui
t asc
orba
te (m
g/10
0 g
FW)
35S-A
tVT2
35S-S
tVTC2A
PAT-StV
TC2A
Empty ve
ctor
WT
35S-A
tVT5
35S-S
tVTC2B
PAT-StV
TC2B
Empty ve
ctor
WT
0
0.5
1
1.5
2 Potato
Tube
r Asc
orba
te (m
g/g
DW)
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 GT C AC T
C
Non-ATG Start Codons
CRICOS No. 00213JLaing 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
Num
ber o
f mat
chin
g se
quen
ces
B
0 500 1000 1500 20000
20
40
Distance along GGP in base pairs
RN
Ase
qR
ibos
ome
prof
iling
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
0:24 25-50 50:74 75:99 100:124 125:149 150:174 175:1990
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
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 cropsPlant 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. LaingSean BulleyRoy StoryMarcela Martinez-Sanchez
Richard C. Macknight Matthew A. W. Chisnall Chris M. Brown
Sally RodenMeredith Crisp