Protein-RNA crosslinking techniques and applications Flashcards
1
Q
What is an RNP? (2)
A
- Ribonucleoprotein
- I.e. RNA is usually bound to lots of proteins so need in vivo approaches to identify which RNAs a protein is interacting with and where it interacts
2
Q
What kind of information can you get from RNA-protein crosslinking analysis?
A
Wide breadth and depth because you can see globally what RNAs a protein interacts with as well as the exact position of each interaction
3
Q
What are the general steps of RNA-protein crosslinking techniques? (5)
A
- Crosslinking the protein and RNA usually with UV
- Purification of RNPs
- cDNA synthesis
- Next generation sequencing of isolated cDNA
- Mapping of sequence reads with nucleotide precision
4
Q
What are the predominant 2 techniques for protein-RNA crosslinking?
A
- CRAC
- iCLIP
5
Q
What is CRAC?
A
Crosslinking and cDNA analysis
6
Q
What are the steps of CRAC? (13)
A
- Starts with genetic engineering of an N or C-terminally affinity-tagged bait protein of interest (e.g. protein A and His tags) with a TEV cleavage site between them which is important for protein isolation later
- Grow cell culture and expose to UV radiation (254nm) to crosslink all proteins and all RNA including bait protein of interest and its bound RNAs
- Prepare cell extract
- Pull out protein of interest with its bound RNAs with an antibody which is specific to the affinity tag (protein A)
- Extraction step is under high salt conditions to prevent protein-protein crosslinks, just protein-RNA
- Cleave at TEV site to isolate protein of interest + bound RNA
- Partial RNase treatment to make RNA fragments for sequencing, any RNA directly bound to and protected by the protein won’t get digested
- 2nd purification step under denaturing conditions to get rid of any additional proteins bound using His tag, doesn’t affect the covalent bond between bait protein and RNA
- Attach 5’ and 3’ linker regions of known sequence to the RNA fragments, 5’ radiolabel to visualise
- Run on SDS-PAGE and physically extract
- Proteinase digest to get just the RNA fragment with the linkers
- Reverse transcription to make a cDNA copy
- PCR to amplify the cDNA and then sequence
7
Q
What is iCLIP?
A
Individual nucleotide resolution UV crosslinking and immunoprecipitation
8
Q
What are the steps of iCLIP? (11)
A
- Largely same principles as CRAC but a few key differences
- Don’t tag protein of interest start with UV irradiate cells to crosslink all protein and RNA
- Prepare cell extract and partial RNase treatment
- Isolate protein of interest with an antibody specific to the bait protein with its bound short RNA fragments
- No second purification step
- Add 3’ linker to the RNA fragments and 5’ label
- Run on SDS-PAGE and physically extract
- Proteinase treatment
- Reverse transcription using a cleavable oligo to make cDNA copy
- Ligation to make cDNA circular then cleave the oligo which produces a linear fragment with unique known sequences on the 5’ and 3’ ends
- PCR and next gen sequencing
9
Q
What is the wavelength of UV radiation?
A
254nm
10
Q
What are the major differences between CRAC and iCLIP? (3)
A
- CRAC uses a tagged protein but iCLIP requires an antibody to the bait protein
- CRAC has 2 purification steps, one of which is under denaturing conditions
- CRAC uses separate 5’ and 3’ linkers but iCLIP uses a single 3’ linker, cleavable oligo and circularisation step
11
Q
What are the pros and cons of the differences between CRAC and iCLIP? (3)
A
- iCLIP is challenging if you don’t have a good specific antibody for the bait protein, however sometimes tagging proteins for CRAC disrupts the function
- Tagging in CRAC allows 2 purifications which increases signal over noise and allows modifications of the approach however this decreases overall recovery
- Circularisation isn’t very efficient in iCLIP but not much difference between this part of the method
12
Q
What are the similarities between CRAC and iCLIP? (3)
A
- Both can give transcriptome wide interaction profiles of a single protein
- Both provide single nucleotide resolution of where protein binds to the RNA
- Output is the same and allows for similar analysis
13
Q
What is the workflow of RNA sequence analysis? (5)
A
- Start with raw sequence reads
- Remove adapter sequences
- Remove amplification duplicates
- Map to whole genome or specific RNA species
- Actual sites of crosslinking can often be identified as there are errors/gaps that arise in the reverse transcriptase step due to the presence of the crosslinked amino acid
14
Q
What is snoRNA? (2)
A
- Small nucleolar RNA
- Involved in ribosome synthesis and mediate modification of rRNA
15
Q
How were U3 snoRNP interaction sites on a specific RNA mapped? (4)
A
- U3 snoRNA is known to associate with multiple proteins to form U3 snoRNP
- CRAC was used to map the interactions on U3 snoRNA e.g. Rrp9
- Nucleotide deletions/substitutions in the sequence reads arising during reverse transcription are indicative of protein crosslinking at this position (single nucleotide resolution)
- Can map the position of the hits along the sequence and map the interaction sites on the secondary/tertiary structure of the RNA
16
Q
What was discovered about hnRNPC from iCLIP? (6)
A
- Nascent transcripts associate with hnRNPs
- Looking at the role of hnRNPC in splicing regulation
- Observed the association of hnRNPC with both exons and introns (pre-mRNA) and the association with alternate forms of mRNA
- Then did metagene analysis which represents average binding pattern of a protein across all genes in the cell using a normalised pre-mRNA
- Identified that hnRNPC generally assembled on both exons and introns but was excluded from splice sites
- Also identify propensity for specific sequences i.e. hnRNPC recognises uridine tracts
17
Q
What is a nascent transcript?
A
Newly synthesised RNA molecule still associated with RNA polymerase (unprocessed)
18
Q
What are hnRNPs?
A
Heterogeneous nuclear ribonucleoproteins
19
Q
What are the general applications of iCLIP and CRAC? (3)
A
- Can identify all RNA species that a specific protein interacts with
- Can identify where on the RNA a protein binds (single nucleotide precision)
- Can identify patterns of binding
20
Q
What are examples of modifications of protein-RNA crosslinking approaches? (2)
A
- Split CRAC
- PAR-CLIP
21
Q
What is split CRAC?
A
Mapping the binding patterns of different domains of the same protein
22
Q
What is PAR-CLIP (2)
A
- Photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation
- More sensitive mapping technique to map low abundance or transient interactions using a uridine analogue
23
Q
How does split CRAC work? (3)
A
- In normal CRAC there are 2 affinity tags separated by the cleavage site but split CRAC repositions the tags and cleavage sites on different domains of the bait protein to determine specific interactions of the domains
- Uses an additional preScission protease (PP) cleavage site
- Largely the same process as normal CRAC but purification steps allow isolation of different protein domains
24
Q
What is an example of split CRAC? (5)
A
- Identifying which RNAs interact with the exo and endonuclease domains of Rrp44
- Had full length Rrp44 with His-TEV-ProtA tag for whole protein interaction
- Second version had endo domain tagged with His-PP and exo domain tagged with TEC-ProtA
- Third version had exo domain tagged with His-TEV-ProtA but exo and endo domains where separated by additional PP site
- Can identify which protein domains are interacting with specific RNAs
25
What is Rrp44? (4)
- Ribonuclease part of the exosome complex
- Has endo and exonuclease activity
- Endo activity encoded by the PIN domain
- Exo activity encoded by the RNB domain
26
What is an example of PAR-CLIP? (6)
- Grow cells in media which allows metabolic labelling i.e. 4 thiouridine (4sU) analogue
- Label is incorporated into newly synthesised RNAs
- Then irradiate cells at 365nm (not standard UV wavelength) to induce crosslinking between incorporated 4sU and its associated proteins
- Then immunoprecipitate protein of interest, prepare cDNA library and sequence like normal iCLIP
- Produces 100 to 1000-fold increase in crosslinked RNA
- Can more accurately identify transient or low abundance interactions
