Reconstitution of archaeal H/ACA sRNPs and test of their activity

Conditions for the reconstitution of archaeal sRNPs active in RNA-guided posttranscriptional modification of RNAs (2'-O-methylation or pseudouridylation) were recently developed. This has opened a vast field of research on structure-function relationships of the sRNP components. We present here an efficient method for in vitro reconstitution of H/ACA sRNPs with an active RNA-guided pseudouridylation activity. They are assembled from an in vitro transcribed H/ACA sRNA with recombinant L7Ae, aCBF5, aNOP10, and aGAR1 proteins. The protocol to test the activity of the assembled particles by the method of the nearest neighbor is also described. The combination of in vitro assembly of H/ACA sRNPs together with time course analysis of pseudouridine formation in the target RNA is useful for structure-function analysis of H/ACA sRNPs and also to identify the target sites of H/ACA sRNAs discovered by computer analysis of archaeal genomes. Furthermore, this efficient in vitro pseudouridylation system can be used for generation of pseudouridine residues at defined positions in RNAs.