DNA-RNA crosslinking techniques (lect 8)

Question 1

Differences between CRAC and iCLIP

Answer 1

Purification steps
-CRAC uses an affinity tag whilst iCLIP uses antibodies
-CRAC has a second purification step whilst iCLIP only have one

Linker ligations
-in CRAC, linkers are added to 3’ and 5’ whilst in iCLIP, they are only added to 3’ (circularisation and linearisation steps ensure seqs at 3’ and 5’ are known)

Question 2

Why might using an affinity tag for purification in CRAC be disadvantageous?

Answer 2

addition of tag may make protein non-functional

Question 3

Why might using an antibody for purification in CRAC be disadvantageous?

Answer 3

specific antibody is needed

Question 4

What are the advantages and disadvantages of having a second purification in CRAC?

Answer 4

Advantages
-better signal over noise
-approach can be modified
Disadvantages
-worse recovery

Question 5

What do DNA-RNA crosslinking techniques (eg. CRAC and iCLIP) show?

Answer 5

Protein interaction sites on a specific RNA

RNA interactions on a specific protein
-patterns in interactions (eg. nt or positional preferences)
-transcriptome-wide info
-info about role of RNA

Question 6

What did iCLIP analysis of hnRNPC on CD55 pre-mRNA show?

Answer 6

preferences of hnRNPCs for introns and exons (and not splice sites) and for uridine tracts