Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs

Abstract

H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.

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

“…RGG domains are frequent in RNAbinding proteins and they play accessory roles in RNA binding (Thandapani et al 2013). The RGG domains of yeast Gar1 have been found to facilitate pseudouridylation of the bound substrate RNA and to inhibit its release, raising the possibility that eukaryotic Gar1 may directly interact with target RNAs though its RGG domains (Trucks et al 2021).…”

Small but strong: the emerging role of small nucleolar RNA in cardiovascular diseases

“…snoRNAs can be categorized into two main structural classes based on their structure, conserved sequence, and rRNA modification functions ( Huang W et al, 2022 ), C/D Box snoRNA ( Massenet et al, 2017 ; Yu et al, 2018 ; Baldini et al, 2021 ) and H/ACA Box snoRNA ( Stepanov et al, 2015 ; Caton et al, 2018 ; Trucks et al, 2021 ). C/D Box snoRNA and H/ACA Box snoRNA families predominate and have been exhaustively researched, whereas limited literature exist on small Cajal body (CB)-specific RNAs (scaRNAs) ( Deryusheva and Gall, 2013 ; Beneventi et al, 2021 ) and orphan snoRNAs ( Kocher et al, 2021 ; Xu et al, 2021 ).…”

“…H/ACA Box snoRNAs (120–140 nucleotides) are characterised by the hallmark ‘hairpin-hinge-hairpin-tail’ secondary structure, which consists of two hairpin domains connected by single-stranded (hinge) and 3′-terminus (tail) regions ( Stepanov et al, 2015 ). The H Box (ANANNA) and ACA Box are located close to the hairpin in the hinge and tail regions, respectively ( Figure 1C ) ( Trucks et al, 2021 ). The H/ACA Box snoRNA antisense region is composed of two short (9–16 nucleotides) sequences located in the internal loops of the 5′ and/or 3′ hairpins.…”

iSnoDi-LSGT: identifying snoRNA-disease associations based on local similarity constraint and global topological constraint

“…With the development of computer technology and experimental approaches, more and more snoRNAs are identified and their functions revealed, such as gene expression, RNA synthesis, transport, and nucleotide modifications, etc. ( Lowe and Eddy 1999 ; Huttenhofer et al 2001 ; Zhao et al 2017 ; Dong et al 2020 ; Hasler et al 2020 , 2021 ; Yang et al 2020 ; Deryusheva et al 2021 ; Shi et al 2021 ; Trucks et al 2021 ).…”