Interaktionen von RNAs und Proteinen

Transcription

1 Sonja Prohaska Computational EvoDevo Universitaet Leipzig June 9, 2015

2 Studying RNA-protein interactions Given: target protein known to bind to RNA problem: find binding partners and binding sites experimental high-throughput techniques based on immunoprecipitation (IP) use antibodies against target protein RIP-seq: full RNAs associated with protein iclip and HITS-CLIP: RNA binding sites of proteins PAR-CLIP: nucleotide resolution RNA-protein interactions

3 Find RNAs bound by target protein RIP = RNA Immuno-Precipitation key feature: no crosslinking problem: may yield indirect interactions

4 Analysis of RIP-seq data what we want: full sequences of the protein-bound RNAs (all!) what we get: reads task for the bioinformatician as in transcriptome sequencing (polish reads: remove adapters and barcodes) map reads to the genome/transcriptome (allow for indels and spliced-reads) cluster reads compare location of clusters to genome annotation identify the bound RNA (if possible) difficulty: alternative transcripts

5 CLIP-seq methods general idea CLIP = Cross-Linking Immuno-Precipitation UV light is used to crosslink RNA and protein in vivo stringent purification: immunoprecipitation, SDS-PAGE, transfer to nitrocellulose proteinase K digests protein but leaves 1-2 amino acids at the UV crosslinked sites reverse transcriptase (RT) makes cdna cdna often truncates at UV crosslinked sites... methods differ here... sequencing bioinformatic analysis

6 CLIP-seq methods general idea Basic principle of all CLIP methods

7 Find RNA binding sites of target protein HITS-CLIP = HIgh-Throughput Sequencing of RNA isolated by Cross-Linking Immuno-Precipitation 254nm UV crosslinking in vivo cell lysis requires 3 - and 5 -ligated adapters (for amplification) revers transcriptase (RT) makes cdna stalling of the RT at UV crosslink sites no amplification (5 -adapter missing) read through at UV crosslink sites induces mutations key feature: crosslink-induced mutation site (CIMS) Problem: high sequencing depths required Problem: destinguish CIMS from technical errors

8 Find RNA binding sites of target protein HITS-CLIP = HIgh-Throughput Sequencing of RNA isolated by Cross-Linking Immuno-Precipitation key feature: crosslink-induced mutation site (CIMS)

9 Find RNA binding sites of target protein iclip = individual-nucleotide resolution CLIP key feature: revers transcription terminates at crosslinks key insight: sites of trancation are sites of crosslinking

10 just another graphics for iclip

11 Single nucleotide contacts of target protein and RNA PAR-CLIP = Photo-Activated RNA CLIP key feature: photo-activatable nucleotides crosslinks key insight: nucleotide transitions indicate crosslinks

12 Single nucleotide contacts of target protein and RNA PAR-CLIP = Photo-Activated RNA CLIP photo-activated nucleotides 4SU and 6SG fed to cells, incorporated in nascent RNA reverse transcriptase inserts G opposite of 4SU = T C transition reverse transcriptase inserts T opposite of 6SG = G A transition bioinformatics: search read clusters with significantly high T > C mismatch frequency RNA U U A G G RNA U U A G G A A T C C A A T C C RNA U 4SU A G G RNA U U A 6SG G A G T C C A A T T C U C A G G U U A A G A G T C C A A T T C

13 CLIP-seq data What do we get? RBP HITS CLIP crosslinking sites errors cdna iclip PAR CLIP HITS-CLIP-seq: nucleotide substitutions around the BS iclip: cdna/read ends at crosslinked nucleotides PAR-CLIP: particular substitutions at crosslinked nucleotides

14 Limitations

15 Protein-Protein Interactions

16 Protein-Protein Interactions stable interactions - oligomers oligomerization, dimerization domains, leucine-zipper covalent-bonds, hydrophobic contacts, salt bridges, disulphid briges, electon sharing transient interactions hydrophobic contacts, Van der Waals forces, hydrogen bonds define interaction interfaces/surfaces rather than specific domains known domains: SH2, SH3, PDZ, SAM

17 Factors regulting Protein-Protein Interactions protein concentrations, which in turn are affected by expression levels and degradation rates presence of other proteins, nucleic acids, and ions electric fields around proteins occurrence of covalent modifications

18 SH2 domain Src Homology 2 2 α-helices and 7 β strands known to identify a sequence of 3-6 aa high affinity to phosphorylated tyrosine function signaling about 100 human proteins

19 SAM domain Sterile Alpha Motif around 70 aa small five-helix bundle seems to possess the ability to bind RNA two large interfaces, form dimers small group of genes

20 PDZ domain aa 5 beta-sheets, helices binds to C-terminus of binding partner adding a β-strand to the β-sheet often multiple PDZ per protein increasing specificity 260 PDZ in 180 human genes

21 Example: Histones of Archaea left: archeal histone dimer, aa in DNA contact (blue), aa in tetramer-formation (orange); right: archeal histone tetramer with DNA; H3 cyan; H4 red; histone fold: helix-loop-helix-loop-helix Archaeal histones can form homo- and heterodimers. Eukaryotic histones can only form hetero-dimers.

22 Protein Complex Graph

23 Yeast two-hybrid (Y2H)