Master thesis @ BioBix

What the f*#@$k are we working on (aka, TOPICS):

•Measuring proteins @ the mRNA level

•Does translation start at ATG?

•Small stuff does matter…

•Knock out those proteins in the translation machinery!

•Epigenetics on RNA?

Measuring proteins @ the mRNA levelMass spectrometry NGS: RNA-seq

Measuring proteins @ the mRNA level

- Harringtonine - Lactimidomycin (LTM)- Puromycin

causes ribosome accumulation at translation initiation site (TIS)

(Ingolia N. et al., Cell, 2011)

(Lee S. et al., PNAS, 2012)

(Fritch C. et al., Gen. Research, 2012)

(Ingolia N. et al., Nature Protocols, 2012)

(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

(3)Quantitative conversion of the protected RNA fragments into a DNA library

(4)That can be analyzed by deep sequencing

(1)

(2)

(3) (4)

Measuring proteins @ the mRNA level

(1) 65% of transcripts contain more than 1 detectable TIS (mESC) (16% ≥ 4) --> Complexity of Proteome

(2) N-terminal truncations and/or extensionsAlternate reading frames (internal out-of-frame), alternative splice

isoformsuORFs (regulation downstream initiation)

(3) Often @ near-cognate initiation sites

(Ingolia N. et al., Cell, 2011)

n =13.454

Does translation start at ATG?

Translatome complexity!

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 AAEnod40: 2 peptides: 12 and 24 AATarsal-less/pri: 4 peptides: 11 and 32 AA Sarcolamban/Sarcolipin: 1 peptide: 29 AA

Small stuff does matter…

Mus musculus(common house mouse)

Small stuff does matter…

Homo Sapiens(common Human)

Knock out those proteins in the translation machinery!

Knock out:eukaryotic translation initiation factors

acetyl transferases

Where can you help out?

•Bioinformatics:

1. tool/pipeline development

2. data analysis/integration (computational genomics)

3. algorithm development

•Technological/Practical aspects: NGS and MS

4. xxx-seq, (replace xxx with MBD/RNA/RIBO/RRBS/CHIP), Mass Spectrometry, …

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

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

Example: Bioinformatics, tool/pipeline development

(International) collaborations:

•VIB (Medical Proteomics Group: ribosome profiling and MS)

•VITO (Mass Spectrometry: small stuff)

•NXTGNT (UGent sequencing facility)

•Asklepios study investigators group

•Weissman lab, Fenyö lab (US: ribosome profiling, cancer proteogenomics, MS)

• 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

More info @: