Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal antisense sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5 '-UN-3 ', including the 5 ' substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (psi) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (psi psi or psi N psi) in the 28S (psi 3747/psi 3749), 18S (psi 1045/psi 1046), and U2 (psi 43/psi 44 and psi 89/psi 91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal psi s, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs. In this study, Jady et al. use genetic depletion and restoration and RNA mutational analyses to demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines in the 28S, 18S, and U2 RNAs, respectively. Their findings show that the dual guide loops form alternative base-pairing interactions with their target RNAs in order to position two substrate Us for pseudouridylation, and this structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy.

Automated summary

This study demonstrates that at least four human H/ACA RNAs have dual pseudouridylation loops, supporting the synthesis of two consecutive pseudouridines in specific human RNAs. The dual guide loops form alternative base-pairing interactions with their target RNAs, allowing the positioning of two substrate Us for pseudouridylation. This structural flexibility provides versatility for RNA-directed pseudouridylation without compromising efficiency and accuracy.