Hi

Yes, you can but under these conditions  the complex might be falling apart.  I suggest in vivo cross-linking...when you do it  in vitro things fall apart and non-specific evens can take place...this is why CLIP etc...I done on live cells...

Hi, this should work but there is number of reasons why is better to UV cross-link in-vivo, two most important:

1. In vivo cross-linking is able to recover very transient interactions that exist for a very short time in actively growing cells. I.e. final cleavage in ribosome biogenesis is catalyzed by Nob1. Nob1 interaction with cleavage site is so short that was visible only when cross-linked in vivo, and not present after in vitro cross-linking. Moreover, Lebaron et al Mol Cell 2012 shows differences between in vivo and in vitro RNA-protein cross-linking

2. There is a higher chance to recover interactions that do not exist. That was nicely shown by Helwak et al Cell 2013 where human AGO1 was used to recover RNA-RNA hybrids. When human lysate was mixed with yeast lysate authors recovered human-yeast RNA hybrids.

The first idea to cross-link frozen powder is much better if you have no possibility to cross-link in vivo.

Thank you Shula and Tomasz. You are tight and in vitro cross linking has its down sides. We cannot do in vivo cross linking because we harvest cells at small intervals of 3 min. The cross linking procedure is very long relative to these intervals.

 My idea was to break the cells in buckets that are frozen with liquid nitrogen  and keep the powder extremely cold (on dry ice) while UV irradiating it. I saw it in a CLIP protocol that I have read. I suspect that doing it that way keeps all the in vivo interactions intact. I hope that someone who has tried it will give us a feedback.  What do you say?

Dear Mordechai,

We've just made preorder for http://www.vari-x-link.com/ which allows for extremly fast UV cross-linking. Machine should arrive at the beginning of next year.

Today it's worth to give a shot with frozen powder. Control to compare in vivo and in vitro cross-linking for zero time point is needed and should solve your doubts.

Dear Tomasz. The control you suggested is brilliant!! Thank you. Thank you also for the link. 

Hi Motti,

I've asked our collaborator Markus Landthaler for his advise (he developed PAR-CLIP and doing CLIP routinely). He suggests to do the crosslink using the powder. The lysed cells are probably not uniformly crosslinked.

I agree with Tomasz's control, but I will also add the last time point as well.

I few other (positive) controls to consider:

1. poly-G or flavivirus Xrn1-resistant RNA elements (xrRNAs) - might have a chance to get "stuck" with Xrn1 for longer times than other RNAs that associate with it.

2. use the D208A mutant that we know binds but does not degrade mRNA. 

Hi Gal. I am so glad to hear this. I also think that the powder might even be better because once the cells are broken, the natural cell to cell variation is also broken. Could you please ask Markus for his detailed protocol?

The Poly(G) control is a great idea for a number of reasons. In combination with the D208A, it can highlight binding of decay intermediates.

Thank you very much!

Motti