interaktionen und modifikationen von rnas und proteinen - … · 2018. 5. 7. · synthesis...
TRANSCRIPT
![Page 1: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/1.jpg)
Interaktionen und Modifikationen von RNAs undProteinen
(Modul 10-202-2208; Spezialvorlesung)
Jorg Fallmann
Institute for BioinformaticsUniversity of Leipzig
04.05.2018
1 / 29
![Page 2: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/2.jpg)
Central Dogma of Gene Expression
DNA → RNA → protein
• only about 2% of human DNA is protein codingabout 80% is transcribed
1 / 29
![Page 3: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/3.jpg)
With great power comes great responsibility
A key mechanism for the survival of each cell and as aconsequence whole organisms, is tight and correct regulationof gene expression
This includes localization, processing and turnover of all formsof nucleic acids (and proteins) in a cell
With growing complexity of organisms, so grows thecomplexity of regulatory mechanisms
2 / 29
![Page 4: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/4.jpg)
With great power comes great responsibility
A key mechanism for the survival of each cell and as aconsequence whole organisms, is tight and correct regulationof gene expression
This includes localization, processing and turnover of all formsof nucleic acids (and proteins) in a cell
With growing complexity of organisms, so grows thecomplexity of regulatory mechanisms
2 / 29
![Page 5: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/5.jpg)
With great power comes great responsibility
A key mechanism for the survival of each cell and as aconsequence whole organisms, is tight and correct regulationof gene expression
This includes localization, processing and turnover of all formsof nucleic acids (and proteins) in a cell
With growing complexity of organisms, so grows thecomplexity of regulatory mechanisms
2 / 29
![Page 6: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/6.jpg)
Gene Expression Regulation Simple
3 / 29
Tran
scri
pti
on
al con
trol
Histones, DNA and DNA binding proteins
![Page 7: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/7.jpg)
Gene Expression Regulation Simple
3 / 29
Tran
scri
pti
on
al con
trol
Histones, DNA and DNA binding proteins
Transcription
5' UTR CDS 3' UTR poly-A tail
RNA and RNA binding proteins
Post-
tran
scri
pti
on
al con
trol
DecayTranslation
![Page 8: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/8.jpg)
RNA cycle of life
Synthesis
RNA-polymerase transcription from DNA template
Processing
Via various complexesde-/capping, de-/adenylation, splicing, modification, ...
Final un-/stable RNA molecule
Available RNA depending on ratiosynthesisdecay
4 / 29
![Page 9: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/9.jpg)
RNA cycle of life
Synthesis
RNA-polymerase transcription from DNA template
From here on post-transcriptional
Processing
Via various complexesde-/capping, de-/adenylation, splicing, modification, ...
Final un-/stable RNA molecule
Available RNA depending on ratiosynthesisdecay
4 / 29
![Page 10: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/10.jpg)
RNA cycle of life
Synthesis
RNA-polymerase transcription from DNA template
Processing
Via various complexesde-/capping, de-/adenylation, splicing, modification, ...
Final un-/stable RNA molecule
Available RNA depending on ratiosynthesisdecay
4 / 29
![Page 11: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/11.jpg)
RNA cycle of life
Synthesis
RNA-polymerase transcription from DNA template
Processing
Via various complexesde-/capping, de-/adenylation, splicing, modification, ...
Final un-/stable RNA molecule
Available RNA depending on ratiosynthesisdecay
4 / 29
![Page 12: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/12.jpg)
RNA cycle of life
Synthesis
RNA-polymerase transcription from DNA template
Processing
Via various complexesde-/capping, de-/adenylation, splicing, modification, ...
Final un-/stable RNA molecule
Available RNA depending on ratiosynthesisdecay
4 / 29
![Page 13: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/13.jpg)
Affects all sort of RNA (mRNA, tRNA, miRNA, lncRNA, ...)
Major part of RNA half-life control is performed directly by
proteins or protein-complexes
5 / 29
![Page 14: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/14.jpg)
Affects all sort of RNA (mRNA, tRNA, miRNA, lncRNA, ...)
Major part of RNA half-life control is performed directly by
proteins or protein-complexes
5 / 29
![Page 15: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/15.jpg)
Affects all sort of RNA (mRNA, tRNA, miRNA, lncRNA, ...)
Major part of RNA half-life control is performed directly by
proteins or protein-complexes
5 / 29
![Page 16: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/16.jpg)
Interaction as the key
For regulation to take place, there has to be interaction
This means direct contact between RNA and protein
Or the lack thereof
6 / 29
![Page 17: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/17.jpg)
Interaction as the key
For regulation to take place, there has to be interaction
This means direct contact between RNA and protein
Or the lack thereof
6 / 29
![Page 18: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/18.jpg)
RNA-protein interactions (RPIs)
Interplay between ribo-nucleic acids (RNA), either coding(mRNA) or non-coding (ncRNA), and RNA binding proteins(RBPs)
Extreme versatility of RNA molecules (sequence and structuralfeatures) is reflected by an equal complexity of RNA bindingdomains and binding preferences of proteins
Remember the many structures for single molecule of RNA
7 / 29
![Page 19: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/19.jpg)
RNA-protein interactions (RPIs)
Interplay between ribo-nucleic acids (RNA), either coding(mRNA) or non-coding (ncRNA), and RNA binding proteins(RBPs)
Extreme versatility of RNA molecules (sequence and structuralfeatures) is reflected by an equal complexity of RNA bindingdomains and binding preferences of proteins
Remember the many structures for single molecule of RNA
7 / 29
![Page 20: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/20.jpg)
RNA-protein interactions (RPIs)
Interplay between ribo-nucleic acids (RNA), either coding(mRNA) or non-coding (ncRNA), and RNA binding proteins(RBPs)
Extreme versatility of RNA molecules (sequence and structuralfeatures) is reflected by an equal complexity of RNA bindingdomains and binding preferences of proteins
Remember the many structures for single molecule of RNA
7 / 29
![Page 21: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/21.jpg)
RNA binding proteins (RBPs)
Hundreds of RNA binding proteins (RBPs) have been shownto be involved in virtually all aspects of (post-transcriptional)gene expression regulation
A manually curated collection of more than 1.500 RBPs inhuman highlights their vast number and potential forinteraction and regulation
8 / 29
![Page 22: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/22.jpg)
RNA binding proteins (RBPs)
Hundreds of RNA binding proteins (RBPs) have been shownto be involved in virtually all aspects of (post-transcriptional)gene expression regulation
A manually curated collection of more than 1.500 RBPs inhuman highlights their vast number and potential forinteraction and regulation
8 / 29
![Page 23: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/23.jpg)
RNA binding proteins (RBPs) II
Regulation is usually initiated by direct interaction betweenRBP and target RNA, requiring more or less specific sequencemotifs and accessible binding sites
Many of the known RBPs seem to prefer single strandedbinding regions, although some have been shown to interactwith structured RNA sites
9 / 29
![Page 24: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/24.jpg)
RNA binding proteins (RBPs) II
Regulation is usually initiated by direct interaction betweenRBP and target RNA, requiring more or less specific sequencemotifs and accessible binding sites
Many of the known RBPs seem to prefer single strandedbinding regions, although some have been shown to interactwith structured RNA sites
9 / 29
![Page 25: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/25.jpg)
Binding RNA vs. binding DNA
Feature RNA DNA
chemical nature 2’-OH 2’-desoxy
sequence U T
base paring rule U-A & U-G only T-A
structure stem-loop structures long double helix
helix geometry A-form usually B-form
function information processing information storage
10 / 29
![Page 26: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/26.jpg)
B-form and A-form double-helix
overall shape axis position
Same nucleotide sequences and Same cWW(cis-Watson-Crick/Watson-Crick) base pairs can form very
different helical structures
11 / 29
![Page 27: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/27.jpg)
RNA-Protein Interactions
Every RNA is in complex with unspecifically or specifically bindingproteins. Togehter they form RiboNucleoProtein Complex (RNPs).
RNAs fold into stem-loop structures→ many different types of interactions
proteins with RNA binding domains (RBDs) bind
single stranded RNA (ssRNA)short motifs in unpaired regionscommonly in sequence specific mannerdouble stranded RNA (dsRNA)paired regions of stemscommonly in sequence unspecific manner
12 / 29
![Page 28: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/28.jpg)
RNA-Protein Interactions
Every RNA is in complex with unspecifically or specifically bindingproteins. Togehter they form RiboNucleoProtein Complex (RNPs).
RNAs fold into stem-loop structures→ many different types of interactions
proteins with RNA binding domains (RBDs) bind
single stranded RNA (ssRNA)short motifs in unpaired regionscommonly in sequence specific manner
double stranded RNA (dsRNA)paired regions of stemscommonly in sequence unspecific manner
12 / 29
![Page 29: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/29.jpg)
RNA-Protein Interactions
Every RNA is in complex with unspecifically or specifically bindingproteins. Togehter they form RiboNucleoProtein Complex (RNPs).
RNAs fold into stem-loop structures→ many different types of interactions
proteins with RNA binding domains (RBDs) bind
single stranded RNA (ssRNA)short motifs in unpaired regionscommonly in sequence specific mannerdouble stranded RNA (dsRNA)paired regions of stemscommonly in sequence unspecific manner
12 / 29
![Page 30: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/30.jpg)
RNA binding domains (RBDs)
The versatility of RBPs makes it hard to predict interactionpartners from amino-acid sequence alone.
Most forms of protein interactions require specific, conserved(tertiary) structures with certain amino-acid content, knownas domains
RBPs contain RNA-binding domains (RBDs)
Although these domains are very specific and employ differentinteraction mechanisms, they can share some features thatenable RNA-protein interactions
13 / 29
![Page 31: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/31.jpg)
RNA binding domains (RBDs)
The versatility of RBPs makes it hard to predict interactionpartners from amino-acid sequence alone.
Most forms of protein interactions require specific, conserved(tertiary) structures with certain amino-acid content, knownas domains
RBPs contain RNA-binding domains (RBDs)
Although these domains are very specific and employ differentinteraction mechanisms, they can share some features thatenable RNA-protein interactions
13 / 29
![Page 32: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/32.jpg)
RNA binding domains (RBDs)
The versatility of RBPs makes it hard to predict interactionpartners from amino-acid sequence alone.
Most forms of protein interactions require specific, conserved(tertiary) structures with certain amino-acid content, knownas domains
RBPs contain RNA-binding domains (RBDs)
Although these domains are very specific and employ differentinteraction mechanisms, they can share some features thatenable RNA-protein interactions
13 / 29
![Page 33: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/33.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 34: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/34.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 35: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/35.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 36: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/36.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 37: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/37.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 38: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/38.jpg)
RNA binding domains (RBDs)
Protein domains can fold independently, and this fold plays acrucial role in the specificity of RNA recognition
Hydrogen bonds with backbone and specific interactionsbetween nucleotides and amino-acids are common forsequence specific binding
Electrostatic interactions and stacking contribute to theaffinity of a protein to RNA
In general, RBDs are 35-90aa in size and interact with a smallstretch of nucleotides (3-5nt)
To increase affinity and specificity often in combination withother RBDs in same protein, highlighting the modularity ofmost proteins
Some RBPs do not contain a canonical RBD
14 / 29
![Page 39: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/39.jpg)
Sequence-specific vs. sequence-unspecific binding
Sequence-specificity can be achieved via two strategies
H-bonds between protein backbone and RNA bases→ highly dependent of protein fold (hydrophobic sidechainslooking towards RNA, almost no intramolecular stacking ofRNA bases, instead intermolecular with sidechains, RNA basesnot exposed to solvent, very rigid and specific scaffold)
H-bonds between aa-sidechains and RNA bases (intramolecularRNA base stacking, RNA bases exposed to solvent)
RBPs often employ a mix of strategies to bind their targets→makes prediction of target sequences challenging
For some RBPs specific sequence preferences are known, forothers they can be guessed from the available RBDs or do notexist
15 / 29
![Page 40: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/40.jpg)
Sequence-specific vs. sequence-unspecific binding
Sequence-specificity can be achieved via two strategies
H-bonds between protein backbone and RNA bases→ highly dependent of protein fold (hydrophobic sidechainslooking towards RNA, almost no intramolecular stacking ofRNA bases, instead intermolecular with sidechains, RNA basesnot exposed to solvent, very rigid and specific scaffold)
H-bonds between aa-sidechains and RNA bases (intramolecularRNA base stacking, RNA bases exposed to solvent)
RBPs often employ a mix of strategies to bind their targets→makes prediction of target sequences challenging
For some RBPs specific sequence preferences are known, forothers they can be guessed from the available RBDs or do notexist
15 / 29
![Page 41: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/41.jpg)
Sequence-specific vs. sequence-unspecific binding
Sequence-specificity can be achieved via two strategies
H-bonds between protein backbone and RNA bases→ highly dependent of protein fold (hydrophobic sidechainslooking towards RNA, almost no intramolecular stacking ofRNA bases, instead intermolecular with sidechains, RNA basesnot exposed to solvent, very rigid and specific scaffold)
H-bonds between aa-sidechains and RNA bases (intramolecularRNA base stacking, RNA bases exposed to solvent)
RBPs often employ a mix of strategies to bind their targets→makes prediction of target sequences challenging
For some RBPs specific sequence preferences are known, forothers they can be guessed from the available RBDs or do notexist
15 / 29
![Page 42: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/42.jpg)
RRM – RNA-recognition motif
• most common
• binds ssRNA
• sequence specific
• 4 anti-parallel β-sheetspacked against 2 α-helices
• 4-6nt interaction
• often in tandems/triplets
• very versatile
RRM in pink, RNA (5nt) green
16 / 29
![Page 43: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/43.jpg)
zinc finger (CCCH/CCHC/C2H2)
• C2H2 bind DNA, CCCH RNA,CCHC viral RNA
• coordinate zinc as commonproperty
• unspecific binding to dsRNA
• specific binding to ssRNA
• RNA bases bulged out
• Intermolecular stacking andH-bond between RNA andbackbone
9 zinc fingers (blue), RNA (8nt) grey
17 / 29
![Page 44: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/44.jpg)
dsRBD – double-stranded RNA Binding Domain• 3 anti-parallelβ-sheets
• α-helices on N- andC-terminus
• unspecific binding todsRNA via α-helicesand loop betweenβ-sheets
• contacts backbone
• sequence-specificbinding of minorgroove (ADAR2)
β-sheets (blue), A-form RNA
18 / 29
![Page 45: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/45.jpg)
Protein interaction elements
Interaction between RNA and proteins depends on both, aprotein domain that recognizes the target RNA, as well asRNA elements that are recognizable by the protein domain.
While protein domains have been studied and characterized indetail, RNA elements crucial for interaction are in generalunderstudied
19 / 29
![Page 46: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/46.jpg)
Protein interaction elements
Interaction between RNA and proteins depends on both, aprotein domain that recognizes the target RNA, as well asRNA elements that are recognizable by the protein domain.
While protein domains have been studied and characterized indetail, RNA elements crucial for interaction are in generalunderstudied
19 / 29
![Page 47: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/47.jpg)
Protein interaction elements on sequence level
Among the most prominent metazoan RNA sequenceelements are AU-rich elements (AREs) and GU- or U- richelements (GU/UREs)
Found in many mRNAs and ncRNAs, throughout all genicregions (UTR, CDS, intron, exon)
Of course there are others (e. g. splice sites, poly-A, ...)
20 / 29
![Page 48: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/48.jpg)
Protein interaction elements on sequence level
Among the most prominent metazoan RNA sequenceelements are AU-rich elements (AREs) and GU- or U- richelements (GU/UREs)
Found in many mRNAs and ncRNAs, throughout all genicregions (UTR, CDS, intron, exon)
Of course there are others (e. g. splice sites, poly-A, ...)
20 / 29
![Page 49: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/49.jpg)
Protein interaction elements on sequence level
Among the most prominent metazoan RNA sequenceelements are AU-rich elements (AREs) and GU- or U- richelements (GU/UREs)
Found in many mRNAs and ncRNAs, throughout all genicregions (UTR, CDS, intron, exon)
Of course there are others (e. g. splice sites, poly-A, ...)
20 / 29
![Page 50: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/50.jpg)
So what about structure?
For the interaction of RNAs and proteins, structure is key
Proteins need structure to form binding domains
RNA structure defines if a binding motif is accessible or not
Sequence elements important for single-stranded RNA bindingproteins, but they require the target RNA sequence to beunpaired or in an accessible structural context like the loopsection of a hairpin loop
21 / 29
![Page 51: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/51.jpg)
So what about structure?
For the interaction of RNAs and proteins, structure is key
Proteins need structure to form binding domains
RNA structure defines if a binding motif is accessible or not
Sequence elements important for single-stranded RNA bindingproteins, but they require the target RNA sequence to beunpaired or in an accessible structural context like the loopsection of a hairpin loop
21 / 29
![Page 52: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/52.jpg)
So what about structure?
For the interaction of RNAs and proteins, structure is key
Proteins need structure to form binding domains
RNA structure defines if a binding motif is accessible or not
Sequence elements important for single-stranded RNA bindingproteins, but they require the target RNA sequence to beunpaired or in an accessible structural context like the loopsection of a hairpin loop
21 / 29
![Page 53: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/53.jpg)
So what about structure?
For the interaction of RNAs and proteins, structure is key
Proteins need structure to form binding domains
RNA structure defines if a binding motif is accessible or not
Sequence elements important for single-stranded RNA bindingproteins, but they require the target RNA sequence to beunpaired or in an accessible structural context like the loopsection of a hairpin loop
21 / 29
![Page 54: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/54.jpg)
So what about structure?
22 / 29
![Page 55: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/55.jpg)
Identifying RNA-protein interactions
RPIs are central part of complex interactome → interplay andmechanisms are not simple to investigate
Interaction is not necessarily functional
Proteins can interact with their target not only at specificsites, but in a probing manner known as diffusional search→ further complicating interaction analysis
23 / 29
![Page 56: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/56.jpg)
Identifying RNA-protein interactions
RPIs are central part of complex interactome → interplay andmechanisms are not simple to investigate
Interaction is not necessarily functional
Proteins can interact with their target not only at specificsites, but in a probing manner known as diffusional search→ further complicating interaction analysis
23 / 29
![Page 57: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/57.jpg)
Identifying RNA-protein interactions
RPIs are central part of complex interactome → interplay andmechanisms are not simple to investigate
Interaction is not necessarily functional
Proteins can interact with their target not only at specificsites, but in a probing manner known as diffusional search→ further complicating interaction analysis
23 / 29
![Page 58: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/58.jpg)
Identifying RNA-protein interactions II
In principle, investigating interactions requires someknowledge of the interaction partners
Need for specific probes, antibodies, cell-types or substrates
24 / 29
![Page 59: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/59.jpg)
Identifying RNA-protein interactions II
In principle, investigating interactions requires someknowledge of the interaction partners
Need for specific probes, antibodies, cell-types or substrates
24 / 29
![Page 60: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/60.jpg)
Experimental Methods
Experimental methods for the characterization of RNA-RBPinteractions can generally be broken down into
in vitro assays, which means free from other interacting factorsand under experimental conditions
in vivo approaches which capture a snapshot of RBP bindingto RNAs at natural expression levels or after induction
25 / 29
![Page 61: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/61.jpg)
in vivo VS in vitro approaches
in vitro approaches
use a synthetic RNA bait to capture RBPs from cellularextracts or vice versa
in vivo approaches
technically more challenging but preserve the context ofcompeting or assisting interactions
26 / 29
![Page 62: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/62.jpg)
in vivo VS in vitro approaches
in vitro approaches
use a synthetic RNA bait to capture RBPs from cellularextracts or vice versa
in vivo approaches
technically more challenging but preserve the context ofcompeting or assisting interactions
26 / 29
![Page 63: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/63.jpg)
RNA vs protein centric methods
RNA-centric methods
Protein-centric methods
27 / 29
![Page 64: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/64.jpg)
RNA vs protein centric methods
RNA-centric methods →Pull down RNA
Protein-centric methods →Pull down protein
27 / 29
![Page 65: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/65.jpg)
RNA vs protein centric methods
RNA-centric methods →Pull down RNA → analyze protein
Protein-centric methods →Pull down protein → analyze RNA
27 / 29
![Page 66: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/66.jpg)
RNA vs protein centric methods
RNA-centric methods
Mass spectrometry (MS) identify RBPs bound to RNAAllow the identification of novel RBP interactionsRBPs for which antibodies are hard to come byRequires the purification of enough protein mass (hard)
Protein-centric methods
27 / 29
![Page 67: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/67.jpg)
RNA vs protein centric methods
RNA-centric methods
Mass spectrometry (MS) identify RBPs bound to RNAAllow the identification of novel RBP interactionsRBPs for which antibodies are hard to come byRequires the purification of enough protein mass (hard)
Protein-centric methods
Co-IP of protein with crosslinked targetsRequire some knowledge about protein and specific antibodiesfor IPCan easily be applied in a high-throughput manner and requirelower amounts of starting material (PCR)
27 / 29
![Page 68: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/68.jpg)
Protein-centric methods
Purification of RNA-RBP complexes via target protein
Specific purification methods for protein in vivoor way to express a tagged version in vitro→ recombinantprotein
Immunoprecipitation (IP) of the protein via specific antibodies
Most commonQuality and specificity of AB has huge impact on reliability
Co-IP’d RNA is reverse transcribed into cDNA
PCR amplification (NOT POSSIBLE WITH PROTEINS!!!)
Detect interaction partners from less starting material
Sequencing
28 / 29
![Page 69: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/69.jpg)
Protein-centric methods
Purification of RNA-RBP complexes via target protein
Specific purification methods for protein in vivoor way to express a tagged version in vitro→ recombinantprotein
Immunoprecipitation (IP) of the protein via specific antibodies
Most commonQuality and specificity of AB has huge impact on reliability
Co-IP’d RNA is reverse transcribed into cDNA
PCR amplification (NOT POSSIBLE WITH PROTEINS!!!)
Detect interaction partners from less starting material
Sequencing
28 / 29
![Page 70: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/70.jpg)
Protein-centric methods
Purification of RNA-RBP complexes via target protein
Specific purification methods for protein in vivoor way to express a tagged version in vitro→ recombinantprotein
Immunoprecipitation (IP) of the protein via specific antibodies
Most commonQuality and specificity of AB has huge impact on reliability
Co-IP’d RNA is reverse transcribed into cDNA
PCR amplification (NOT POSSIBLE WITH PROTEINS!!!)
Detect interaction partners from less starting material
Sequencing
28 / 29
![Page 71: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/71.jpg)
Protein-centric methods
Purification of RNA-RBP complexes via target protein
Specific purification methods for protein in vivoor way to express a tagged version in vitro→ recombinantprotein
Immunoprecipitation (IP) of the protein via specific antibodies
Most commonQuality and specificity of AB has huge impact on reliability
Co-IP’d RNA is reverse transcribed into cDNA
PCR amplification (NOT POSSIBLE WITH PROTEINS!!!)
Detect interaction partners from less starting material
Sequencing
28 / 29
![Page 72: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/72.jpg)
Protein-centric methods
Purification of RNA-RBP complexes via target protein
Specific purification methods for protein in vivoor way to express a tagged version in vitro→ recombinantprotein
Immunoprecipitation (IP) of the protein via specific antibodies
Most commonQuality and specificity of AB has huge impact on reliability
Co-IP’d RNA is reverse transcribed into cDNA
PCR amplification (NOT POSSIBLE WITH PROTEINS!!!)
Detect interaction partners from less starting material
Sequencing
28 / 29
![Page 73: Interaktionen und Modifikationen von RNAs und Proteinen - … · 2018. 5. 7. · Synthesis RNA-polymerase transcription from DNA template From here on post-transcriptional Processing](https://reader034.vdocuments.site/reader034/viewer/2022051904/5ff5fb32aa37b073127e2202/html5/thumbnails/73.jpg)
Protein centric assays
29 / 29