If we use RNase to treat the RNA (pulldown by RNA IP experiment). Can RNA binding protein protect the RBPs binding region on RNAs from RNase degradation?
Yes and no.
Depending on the RNAse, you'll probably protect SOME of the RNA, for SOME time.
This is basically RNA footprinting, which is an established (if slightly old-school) technique. Mild, transient RNAse treatment will preferentially digest exposed RNA and leave bound RNA undigested.
It sounds horrendously fiddly to get to work, though.
This is the basic idea behind CLIP-seq. Including a covalent cross-linking step improves the resistance of the RBP-bound regions to RNase digestion. RBPs are then digested with proteinases, and the liberated RNA fragments are used for sequencing.
This article provides a good overview of the different CLIP-seq approaches:
Article CLIP and complementary methods
John Hildyard John Hardy Lockhart . thank you sir for your nice reply. actually i pulldown mRNA by one of my selected RBP through RNA IP analysis. now i want to know where RBP of my interest bind to the mRNA of few genes i found through RNA IP analysis. for that reason i have to digest the remaining RNA left only the binding region for sequencing.
That's exactly what CLIP-seq will tell you. Check out the article I linked in my previous reply to get an idea of what type of CLIP-seq would best answer your research question.
Abcam has an example protocol for iCLIP-seq that you can look through:
https://www.abcam.com/protocols/uv-cross-linking-and-immunoprecipitation-clip
John Hardy Lockhart Thank you very much sir, I got and will follow your recommendation.
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