Purification and Functional Reconstitution of Box H/ACA Ribonucleoprotein Particles

Pseudouridylation is the most abundant and widespread RNA modification, and it plays an important role in modulating the structure and function of RNA. In eukaryotes and archaea, RNA pseudouridylation is catalyzed largely by box H/ACA ribonucleoproteins (RNPs), a distinct group of RNA-protein complexes each consisting of a unique RNA and four common proteins. The RNA component of the complex serves as a guide that base-pairs with its substrate RNA and specifies the target uridine to be modified. In order to systematically study the function and mechanism of pseudouridylation, it is desirable to have a reconstitution system in which biochemically purified/reconstituted box H/ACA RNPs are capable of introducing pseudouridines into an RNA at any target site. Here, we describe a method for the reconstitution of functional box H/ACA RNPs using designer box H/ACA guide RNAs, which in principle can be adopted to reconstitute other RNA-protein complexes as well.