Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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 (Ψ) 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 (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) 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 Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.

SUBMITTER: Jady BE

PROVIDER: S-EPMC8763049 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Human box H/ACA pseudouridylation guide RNA machinery.

Project description:Pseudouridine, the most abundant modified nucleoside in RNA, is synthesized by posttranscriptional isomerization of uridines. In eukaryotic RNAs, site-specific synthesis of pseudouridines is directed primarily by box H/ACA guide RNAs. In this study, we have identified 61 novel putative pseudouridylation guide RNAs by construction and characterization of a cDNA library of human box H/ACA RNAs. The majority of the new box H/ACA RNAs are predicted to direct pseudouridine synthesis in rRNAs and spliceosomal small nuclear RNAs. We can attribute RNA-directed modification to 79 of the 97 pseudouridylation sites present in the human 18S, 5.8S, and 28S rRNAs and to 11 of the 21 pseudouridines reported for the U1, U2, U4, U5, and U6 spliceosomal RNAs. We have also identified 12 novel box H/ACA RNAs which lack apparent target pseudouridines in rRNAs and small nuclear RNAs. These putative guide RNAs likely function in the pseudouridylation of some other types of cellular RNAs, suggesting that RNA-guided pseudouridylation is more general than assumed before. The genomic organization of the new box H/ACA RNA genes indicates that in human cells, all box H/ACA pseudouridylation guide RNAs are processed from introns of pre-mRNA transcripts which either encode a protein product or lack protein-coding capacity.

| S-EPMC480876 | biostudies-literature

Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.

Project description:H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p-Nop10p-Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.

| S-EPMC5778458 | biostudies-literature

The presence of the ACA box in archaeal H/ACA guide RNAs promotes atypical pseudouridylation.

Project description:Archaea and eukaryotes, in addition to protein-only enzymes, also possess ribonucleoproteins containing an H/ACA guide RNA plus four proteins that produce pseudouridine (Ψ). Although typical conditions for these RNA-guided reactions are known, certain variant conditions allow pseudouridylation. We used mutants of the two stem-loops of the Haloferax volcanii sR-h45 RNA that guides three pseudouridylations in 23S rRNA and their target RNAs to characterize modifications under various atypical conditions. The 5' stem-loop produces Ψ2605 and the 3' stem-loop produces Ψ1940 and Ψ1942. The latter two modifications require unpaired "UVUN" (V = A, C, or G) in the target and ACA box in the guide. Ψ1942 modification requires the presence of U1940 (or Ψ1940). Ψ1940 is not produced in the Ψ1942-containing substrate, suggesting a sequential modification of the two residues. The ACA box of a single stem-loop guide is not required when typically unpaired "UN" is up to 17 bases from its position in the guide, but is needed when the distance increases to 19 bases or the N is paired. However, ANA of the H box of the double stem-loop guide is needed even for the 5' typical pseudouridylation. The most 5' unpaired U in a string of U's is converted to Ψ, and in the absence of an unpaired U, a paired U can also be modified. Certain mutants of the Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of nonconstitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.

| S-EPMC7075261 | biostudies-literature

A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains.

Project description:Site-specific post-transcriptional conversion of uridines to pseudouridine in ribosomal RNAs and small nuclear RNAs (snRNAs) is directed by guide RNAs which possess the conserved box H and ACA sequence elements and fold into the consensus 'hairpin-hinge-hairpin-tail' secondary structure. Here, we describe an unusual mammalian pseudouridylation guide RNA, called U93, that is composed of two tandemly arranged box H/ACA RNA domains. The U93 RNA therefore carries two H and two ACA box motifs, all of which are essential for accumulation of the full-length RNA. The human U93 RNA accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA. Our results lend further support to the notion that modification of the RNA polymerase II-transcribed spliceosomal snRNAs takes place in Cajal bodies.

| S-EPMC135803 | biostudies-literature

An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes.

Project description:U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.

| S-EPMC1370357 | biostudies-other

Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

Project description:H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs.

| S-EPMC6800473 | biostudies-literature

Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation.

Project description:Pseudouridine (Psi) are frequently modified residues in RNA. In Eukarya, their formation is catalyzed by enzymes or by ribonucleoprotein complexes (RNPs) containing H/ACA snoRNAs. H/ACA sRNA and putative ORFs for H/ACA sRNP proteins (L7Ae, aCBF5, aNOP10 and aGAR1) were found in Archaea. Here, by using Pyrococcus abyssi recombinant proteins and an in vitro transcribed P.abyssi H/ACA sRNA, we obtained the first complete in vitro reconstitution of an active H/ACA RNP. Both L7Ae and the aCBF5 RNA:Psi synthase bind directly the sRNA; aCBF5 also interacts directly and independently with aNOP10 and aGAR1. Presence of aCBF5, aNOP10 and a U residue at the pseudouridylation site in the target RNA are required for RNA target recruitment. In agreement, we found that the aCBF5-aNOP10 pair is the minimal set of proteins needed for the formation of a particle active for pseudouridylation. However, particles more efficient in targeted pseudouridylation can be formed with the addition of proteins L7Ae and/or aGAR1. Although necessary for optimal activity, the conserved ACA motif in the sRNA was found to be not essential.

| S-EPMC1142404 | biostudies-literature

Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.

Project description:H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product.

| S-EPMC4551948 | biostudies-literature

Structural dynamics govern substrate recruitment and catalytic turnover in H/ACA RNP pseudouridylation.

Project description:H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.

| S-EPMC8354600 | biostudies-literature

Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines.

Project description:One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNA(Val) containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G.U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

| S-EPMC2816034 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data