XRNAX is a method for the extraction of protein-crosslinked RNA from UV-crosslinked cultured cells. Extracting protein-crosslinked RNA enables unseen proteomic and transcriptomic applications for the interrogation of protein-RNA interfaces or differential RNA binding. Learn here about XRNAX, exemplary applications, and how you can use XRNAX to enhance your protocol.
XRNAX starts out from UV-crosslinked cultured cells, which are lysed in TRIZOL. After addition of chloroform and phase separation free RNA ends up in the aqueous phase, protein in the organic phase but protein-crosslinked RNA gets trapped in the interphase along with chromatin. XRNAX collects the interphase, solubilizes it, and removes DNA through DNase digestion. XRNAX extracts are highly concentrated in protein and RNA, are free of cellular debris and salts and can be taken up in any desired buffer.
RNA in XRNAX extracts can be used as a handle for the co-precipitation of UV-crosslinked protein: Conventional silica spin-columns are employed to free protein-crosslinked RNA from any non-covalent interactions. This allows for the controlled discovery of RNA-binding proteomes when UV-crosslinked cells are compared to non-crosslinked control cells with SILAC. With the same approach, the RNA-binding proteome of differentially treated cells can be compared, thereby assessing differential RNA-binding globally.
XRNAX extracts are free of DNA and cellular debris. This means they can be subjected to high intensity sonication without interference from large molecules other than RNA, yielding RNA fragment sizes from 30-80 nt. These fragments are ideally suited for transcriptomic applications such as CLIP-Seq. RNase fragmentation is a well-known cause for sequencing biases in CLIP-Seq experiments and often requires laborious optimization. Sonicated XRNAX extracts can be used for a CLIP-Seq modification, which refrains from RNA fragmentation through RNase treatment.
XRNAX extracts only contain protein and RNA. This means they can be subjected to the entire arsenal of chemical biology, which was designed for either of the two without interference from cellular components usually present in lysates. For example, proteinase K digested and silica spin-column purified XRNAX extracts can be reacted to NHS-biotin, thereby labeling crosslinking sites.
Moreover, many enzymatic reactions can be performed on XRNAX extracts without interference from metabolites or detergents usually present in lysates. For example, poly(U) polymerase can add biotinylated nucleotides to the 3’ ends of RNA in XRNAX extracts.
Extracts can be alcohol precipitated for quick and efficient cleanup.