Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

Abstract

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site.

The molecular recognition of kink-turn structure by the L7Ae class of proteins

“…Thus, these proteins should recognize a k-loop structure (where the C helix is replaced by a terminal loop) equally well as a k-turn. For example, the L7Ae-family proteins bind the k-loops of the box H/ACA RNA in the assembly of the snoRNP required for pseudouridylation (Rozhdestvensky et al 2003;Hamma and Ferré-D'Amaré 2004;Li and Ye 2006;Youssef et al 2007). …”

Binding and cleavage of CRISPR RNA by Cas6

“…Lead (II)-induced and RNase A cleavage were carried out essentially as described previously (Youssef et al 2007). Briefly, 0.1 pmol of 32 P end-labeled RNA (either 59 or 39) was incubated in the absence (free RNA) or presence of increasing concentrations of Cas6 for 30 min at 65°C-70°C in buffer A (20 mM HEPES-KOH at pH [7.0], 500 mM KCl).…”

“…Reactions were stopped by PCI extraction followed by ethanol precipitation. Alkaline hydrolysis ladders (cleavage after each nucleotide) were generated as described previously (Youssef et al 2007). In each case, precipitated RNAs were resuspended in RNA loading dye (10 M urea, 2 mM EDTA, 0.5% SDS, and 0.02% [w/v] each bromophenol blue and xylene cyanol) and separated on 38 3 30 cm 15% polyacrylamide (acrylamide:bis ratio 19:1) 7 M urea-containing gels.…”

A structural database for k-turn motifs in RNA

“…Further examples have been found in mRNA (Mao et al 1999;Winkler et al 2001;Allmang et al 2002;White et al 2004) and in riboswitches (Montange and Batey 2006;Blouin and Lafontaine 2007;Heppell and Lafontaine 2008). They are very commonly found in C/D and H/ACA guide snoRNAs and in U3 snoRNA species (Kuhn et al 2002;Watkins et al 2002;Bortolin et al 2003;Marmier-Gourrier et al 2003;Rozhdestvensky et al 2003;Hamma and Ferré-D'Amaré 2004;Moore et al 2004;Szewczak et al 2005;Youssef et al 2007). There is also a near-canonical k-turn in a stem-loop of the human U4 snRNA (Vidovic et al 2000;Wozniak et al 2005).…”