This master's thesis project involves studying small regulatory peptides and alternative translation initiation sites using techniques like ribosome profiling, mass spectrometry, and sequencing. Specific topics being investigated include micropeptides, non-ATG translation start sites, nuclear translation, evolutionary epigenetics, imprinted loci identification, and cardiovascular epigenomics. The student is welcome to contribute to tool development for analyzing ribosome profiling and mass spectrometry data, or help with computational analysis and data integration. Potential collaborators mentioned include labs at VIB, VITO, and international institutions studying related topics.

1. Master thesis @ BioBix

2. What the f*#@$k are we working on (aka, TOPICS):
• Small
stuff does matter…
• Does
translation start at ATG?
• Nuclear
translation, ay caramba?
• (R)Evolutionary
• Hunt
epigenetics
for imprinted loci
• Cardiovascular
epigenomics

3. Small stuff does matter…
Canonical bio-active peptides:
-cleaved from precursor
-signal peptide @ N-terminus
-secretory pathway
!
Micropeptides:
-translated directly from sORF
-lacking signal sequence
-released in cytoplasm
Polaris: 3 peptides: 8, 9, 36 AA
Rotundifolia4: 1 peptide: 53 AA
Enod40: 2 peptides: 12 and 24 AA
Tarsal-less/pri: 4 peptides: 11 and 32 AA

4. Small stuff does matter…
Mus musculus
(common house mouse)
Build discovery strategy
Extrapolate strategy
160 Mbp
➡ Examples for
validation are available
➡
model
systems
➡
L2 and L3 type
larvae
➡ Embryonic stages
between 10-16h
➡
3 Gbp
between 8 and
12 days past coitus
(dpc)
➡
samples

5. Small stuff does matter…
(Crappé J. et al., BMC Genomics, 2013)

6. Does translation start at ATG?
Mass spectrometry
NGS: RNA-seq

7. Does translation start at ATG?

8. (1)
(3)
(4)
Does translation start at ATG?
(1) Generation of cell extracts in which ribosomes
have been faithfully halted along the mRNA they
are translating in vivo
(2) Nuclease digestion of RNAs that are not
protected by the ribosome followed by recovery
of the ribosome-protected mRNA fragments
(2)
(3)Quantitative conversion of the protected RNA
fragments into a DNA library
(4)That can be analyzed by deep sequencing
(Ingolia N. et al., Nature Protocols, 2012)
(Ingolia N. et al., Cell, 2011)
- Harringtonine
- Lactimidomycin (LTM)
(Lee S. et al., PNAS, 2012)
(Fritch C. et al., Gen. Research, 2012)
- Puromycin
!
causes ribosome accumulation at translation initiation site (TIS)

9. Does translation start at ATG?
(1) 65% of transcripts contain more than 1 detectable TIS (mESC) (16% ≥ 4)
--> Complexity of Proteome
(2) N-terminal truncations and/or extensions
Alternate reading frames (internal out-of-frame), alternative splice
isoforms
uORFs (regulation downstream initiation)
!
(3) Often @ near-cognate initiation sites
n =13.454
(Ingolia N. et al., Cell, 2011)

10. Nuclear translation, ay caramba?

11. (R)Evolutionary epigenetics

12. (R)Evolutionary epigenetics

13. Hunt for imprinted loci
DNA-(hydroxy)methylatie
ChIP-seq

14. Cardiovascular epigenomics
(TTAGGG)n'

15. Cardiovascular epigenomics

16. Where can you help out?
• Bioinformatics:
1.
tool/pipeline development
2.
data analysis/integration (computational genomics)
3.
algorithm development
• Technological
4.
aspects: NGS and MS
xxx-seq, (replace xxx with MBD/RNA/RIBO/RRBS/
CHIP) or Mass Spectrometry

17. Where can you help out?
Example: Bioinformatics, tool/pipeline development
- Use RIBO-seq translation synthesis products as search space for MS/MS based proteomics/peptidomics
- Construct a user-friendly, robust and fast pipeline to do the conversion
- Both scripting based and implementation in Galaxy-P

18. Where can you help out?
Example: Bioinformatics, tool/pipeline development

19. Example: Bioinformatics, tool/pipeline development
Results:
(1) 45 LC runs resulting in 68.523 MS/MS spectra
(2) Different translation product types
Ribo-seq
N-terminomics
259$
16$
4$
1$
3$
1556$
n =13.454
n =1.835
(Menschaert G. et al., Mol & Cell Prot, 2013)

20. Example: Bioinformatics, tool/pipeline development
(3) Start codon: both cognate and near-cognate
1.0
ACG
0.0
G
TG
G
C
A
A
C
G
C
T
TA
C
G
T
1.0
probability
bits
2.0
T
0.5
G
TG
GA
ACCG
A
GTAC
C
0.0 TGT TC
T
C
A
5
Kozak
mo(f:[A/G]CCatgG[not
T]
Based on new N-term-ext and uORF identifications
T
C
A
5
WebLogo 3.3
WebLogo 3.3
(4) Example: HDGF_MOUSE (n-term-ext, near-cognate start site)
GTG
GTG
TTG
HDGF
(hsa)
stomach&
skin&
spleen&
heart&
kidney&
intes*ne&
lung&
46#
40#kDa#
38#kDa#
36.5#kDa#
HDGF
(mmu)
(V)AAPELASGAGIEAGAAR
(|)||||| || |||||||
(V)AAPELGPGATIEAGAAR
liver&
tes*s&
GCCGTGTGTTGCCCACCGCGCCCGGCCCTGTCCGA
GCGGCGCGCGGGCGCAGACGCCGTGGCTGCCCCGG
AGCTCGCGTCGGGGGCCGGCATCGAGGCGGGGGCC
GCGCGAGGGCCGGAGCGCAGCGGCGCCGCAACCGC
CGCACGCGCAAACTTGGGCTCGCGCTTCCCGGCTC
GGCGCGGAGCCCGGGGCGCCCGCGGCCCCGCCATG
TCGCGATCCAACCGGCAGA…
brain&
uc008ptc.1
muscle&
ATG
32#
HDGF%
(Menschaert G. et al., Mol & Cell Prot, 2013)

21. (International) collaborations:
• VIB
(Medical Proteomics Group: ribosome
profiling and MS)
• VITO
(Mass Spectrometry: small stuff)
• NXTGNT
• Asklepios
(UGent sequencing facility)
study investigators group
• Weissman
lab, Fenyö lab (US: ribosome profiling,
cancer proteogenomics, MS)

22. More info @:
• where are we:
www.biobix.be
general info on the lab
topics online, your input ?
www.nucleotides2networks.be
• where
are we physically:
Building A, 2nd floor, Room 004
symposium, 29th of November:
“Bridging the gap between two omics worlds,
transcriptomics and proteomics”
invitation will follow/be posted on the biobix.be
• Mini