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Local conformations of structural motifs involved in É modification(s) in NMR structures and crystal structures are shown. Unlike U1911 and A1919 in UUU (a), É1911 and A1919 in ÉÉÉ (b) are positioned to form an optimal Watson-Crick base pair. In ÉÉÉ, base moieties of residues from É1915 to A1918 form a continuous-stacking conformation (d), which is not observed in the same region of UUU (c). This continuous base stacking is also observed in the crystal structures of D. radiodurans (PDB ID 1NKW) (e) and E. coli (PDB ID 2I2T) (f) ribosomes.
Source publication
Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered
in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations
of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal...
Contexts in source publication
Context 1
... dihedral angle restraints were obtained from 2D DQF-COSY spectra. In the 2D DQF-COSY spectrum of UUU, three intense cross-peaks of H1'-H2' were assigned to C1914, U1915 and U1917 (Supplementary Figure S4a), indicating a C2'-endo sugar pucker conform- ation. In the 2D DQF-COSY of the ÉÉÉ sample, no H1'- H2' cross-peaks are observed. ...
Context 2
... the 2D DQF-COSY of the ÉÉÉ sample, no H1'- H2' cross-peaks are observed. However, the ÉÉÉ spectrum does have one upfield-shifted cross-peak of weak intensity corresponding to H6-H1' of É1915 (Supplementary Figure S4b). The appearance of this 4 J HH -coupling enables the addition of a dihedral angle (H1'-C1'-C5-C6) constraint of 180 ± 45 , as a coplanar geometry of H6-C6-C5-C1'-H1' is required to maximize the four-bond magnetization transfer, and no strong cross-peak between the H6 and H1' was observed in the corresponding 2D NOESY (D 2 O: 99.96%) spectrum, excluding the possibility of a syn dihedral angle. ...
Context 3
... and A1919 form a canonical Watson-Crick base pair with an additional intraresidue hydrogen bond involving W1911N1H Neighboring the stem-closing base pair (G1910-C1920), U1911 and A1919 in UUU do not form an optimal Watson-Crick base pair (Figure 4a). In the family of 10 UUU NMR structures, the average distances between hydrogen-bond partner pairs UN3H-AN1 and UO4- AN6H are both 2.5 A ˚ (2.5 ± 0.3 A ˚ and 2.5 ± 0.6 A ˚ , re- spectively), whereas the optimal hydrogen-bond distance is 1.8 ...
Context 4
... it is worth noting that the H69 construct with U1911 is 1.0 kcal/mol less stable than a construct with É1911 (37). In contrast, É1911 and A1919 in ÉÉÉ form a canonical Watson-Crick base pair (Figure 4b). The average dis- tances of ÉN3H-AN1 and ÉO2-AN6H are 1.8 ± 0.1 A ˚ and 1.7 ± 0.2 A ˚ , respectively, and the corresponding three-atom geometry is much closer to a linear arrange- ment (170 ± 7 for ÉN3-ÉN3H-AN1 and 164 ± 6 for ÉO2-AN6H-AN6). ...
Context 5
... and W1917 promote local base stacking Two of the three É modifications are clustered in the 3' half of the H69 loop, where distinct conformational dif- ferences are observed between the NMR structures of UUU and ÉÉÉ (Figures 4c and 5d). In ÉÉÉ, residues É1915, A1916, É1917 and A1918 establish a continuous base-stacking motif, whereas two disruptions in base stacking at the U1915-A1916 and U1917-A1918 steps are observed in the UUU RNA. ...
Context 6
... the NMR structure of UUU, the distances between base mass centers of U1915-A1916 ($7 A ˚ ) and U1917-A1918 ($9 A ˚ ) steps are much larger. Because evidence for a C2'-endo sugar pucker of U1915 and U1917 was revealed in the 2D DQF-COSY spectrum of the UUU construct (Supplementary Figure S4a), it is not surprising that these two residues can span much longer distances in UUU compared with the corresponding És in the modified RNA (6,66). Given that the energy of base stacking is approximately proportional to 1/R 6 (i.e. ...
Context 7
... continuous base-stacking motif from É1915 to A1918 was observed in crystal structures of the eubacterial ribo- somal large subunit before (1NKW, D. radiodurans) and after (212T, E. coli) being assembled into complete ribo- somes (Figure 4e and f) (59,60). Because the sequence and É modifications in H69 from D. radiodurans are the same as those in E. coli, the crystal structure of H69 from 1NKW is used to represent the structure of H69 from E. coli before ribosome assembly, which is not currently available. ...
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We present a liquid chromatography-mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-trans...
Citations
... 68 Although the precise mechanism remains unclear, it is thought to involve pseudouridine-induced changes in RNA conformation. 69 Dysregulation of rRNA pseudouridylation primarily affects translational fidelity by altering rRNA structure, which in turn governs numerous biological processes. Given the crucial role of rRNA in ribosome function and its involvement in diverse physiological and pathological processes through RNA epigenetic modifications, further investigation into the role of pseudouridylation in rRNA metabolism is essential. ...
RNA pseudouridylation, a dynamic and reversible post‐transcriptional modification found in diverse RNA species, is crucial for various biological processes, including tRNA homeostasis, tRNA transport, translation initiation regulation, pre‐mRNA splicing, enhancement of mRNA translation, and translational fidelity. Disruption of pseudouridylation impairs cellular homeostasis, contributing to pathological alterations. Recent studies have highlighted its regulatory role in human diseases, particularly in tumourigenesis. Cellular stresses trigger RNA pseudouridylation in organisms, suggesting that pseudouridylation‐mediated epigenetic reprogramming is essential for maintaining cellular viability and responding to stress. This review examines the regulatory mechanisms and pathological implications of pseudouridylation in human diseases, with a focus on its involvement in tumourigenesis. Additionally, it explores the therapeutic potential of targeting pseudouridylation, presenting novel strategies for disease treatment.
Highlights
Methods to detect pseudouridine were introduced from classic mass spectrometry‐based methods to newer approaches such as nanopore‐based technologies and BID sequencing, each with its advantages and limitations.
RNA pseudouridylation is crucial for various biological processes, including tRNA homeostasis, tRNA transport, translation initiation regulation, pre‐mRNA splicing, enhancement of mRNA translation, and translational fidelity.
Increased pseudouridylation is frequently associated with tumour initiation, progression, and poor prognosis, whereas its reduction is predominantly implicated in non‐tumour diseases.
A comprehensive understanding of the inducing factors for RNA pseudouridylation will be essential for elucidating its role in diseases. Such insights can provide robust evidence for how pseudouridylation influences disease progression and offer new avenues for therapeutic strategies targeting pseudouridylation dysregulation.
The therapeutic potential of RNA pseudouridylation in diseases is enormous, including inhibitors targeting pseudouridine synthases, the application of RNA pseudouridylation in RNA therapeutics, and its role as a biological marker.
... However, no effect was observed following changes of a single modification 22,23 . The effect of Ψ modifications on H69 was studied in bacteria and yeast 22,24,25 . H69 interacts with the small subunit rRNA helix 44 (h44) just below the A-site, forming the ribosomal inter-subunit bridge (bridge 2a) 22,24,25 . ...
... The effect of Ψ modifications on H69 was studied in bacteria and yeast 22,24,25 . H69 interacts with the small subunit rRNA helix 44 (h44) just below the A-site, forming the ribosomal inter-subunit bridge (bridge 2a) 22,24,25 . Ψs on the H69 stabilize the helix, enabling the conformational changes of its loop during the dynamic translation process 22,24,25 . ...
... H69 interacts with the small subunit rRNA helix 44 (h44) just below the A-site, forming the ribosomal inter-subunit bridge (bridge 2a) 22,24,25 . Ψs on the H69 stabilize the helix, enabling the conformational changes of its loop during the dynamic translation process 22,24,25 . Most recently, based on the cryo-EM structure of Ψ-free yeast ribosomes, it was proposed that Ψs on H69 promote the interaction with h44, which may facilitate the small subunit head swivel during translation 26 . ...
Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.
... rRNAs bear a large number of pseudouridines, whose function came into light by the discovery of box H/ACA RNAs and their role in rRNA pseudouridylation [48,49]. Pseudouridines are often clustered in functionally vital regions of rRNAs that are important for protein translation and ribosome biogenesis, like peptidyl transferase centre, decoding centre, A-site finger region, helix 69 and even at sites where the ribosomal subunits interact [70][71][72][73] Dyskerin/DKC1, is the primary PUS which mediates rRNA pseudouridylation and is thus critical for ribosome biogenesis [74,75]. DKC1 deficiency is linked with reduced translation fidelity and impaired translation initiation [22, 23]. ...
Cellular RNAs, both coding and noncoding are adorned by > 100 chemical modifications, which impact various facets of RNA metabolism and gene expression. Very often derailments in these modifications are associated with a plethora of human diseases. One of the most oldest of such modification is pseudouridylation of RNA, wherein uridine is converted to a pseudouridine (Ψ) via an isomerization reaction. When discovered, Ψ was referred to as the 'fifth nucleotide' and is chemically distinct from uridine and any other known nucleotides. Experimental evidence accumulated over the past six decades, coupled together with the recent technological advances in pseudouridine detection, suggest the presence of pseudouridine on messenger RNA, as well as on diverse classes of non-coding RNA in human cells. RNA pseudouridylation has widespread effects on cellular RNA metabolism and gene expression, primarily via stabilizing RNA conformations and destabilizing interactions with RNA-binding proteins. However, much remains to be understood about the RNA targets and their recognition by the pseudouridylation machinery, the regulation of RNA pseudouridylation, and its crosstalk with other RNA modifications and gene regulatory processes. In this review, we summarize the mechanism and molecular machinery involved in depositing pseudouridine on target RNAs, molecular functions of RNA pseudouridylation, tools to detect pseudouridines, the role of RNA pseudouridylation in human diseases like cancer, and finally, the potential of pseudouridine to serve as a biomarker and as an attractive therapeutic target.
Graphical abstract
... On one side pseudouridines are known to confer increased rigidity to the phosphodiester backbone of the RNA through effects on base stacking and water coordination. In addition, they are crucial for the formation of specific tertiary structures which may depend on their specific location (stem vs loop) and sequence context (20)(21)(22). Pseudouridine can also confer local conforma tional d ynamics which play important roles in ribosome assembly and maintenance of the ribosomal structural integrity during transla tion. Moreover, pseudourid yla tion a t specific sites can facilita te the further modifica tion of adjacent nucleotides by altering the allosteric arrangement of rRNA ( 22 ). ...
... Pseudouridine can also confer local conforma tional d ynamics which play important roles in ribosome assembly and maintenance of the ribosomal structural integrity during transla tion. Moreover, pseudourid yla tion a t specific sites can facilita te the further modifica tion of adjacent nucleotides by altering the allosteric arrangement of rRNA ( 22 ). ...
RNA modifications are key regulatory factors for several biological and pathological processes. They are abundantly represented on ribosomal RNA (rRNA), where they contribute to regulate ribosomal function in mRNA translation. Altered RNA modification pathways have been linked to tumorigenesis as well as to other human diseases. In this study we quantitatively evaluated the site-specific pseudouridylation pattern in rRNA in breast cancer samples exploiting the RBS-Seq technique involving RNA bisulfite treatment coupled with a new NGS approach. We found a wide variability among patients at different sites. The most dysregulated positions in tumors turned out to be hypermodified with respect to a reference RNA. As for 2′O-methylation level of rRNA modification, we detected variable and stable pseudouridine sites, with the most stable sites being the most evolutionary conserved. We also observed that pseudouridylation levels at specific sites are related to some clinical and bio-pathological tumor features and they are able to distinguish different patient clusters. This study is the first example of the contribution that newly available high-throughput approaches for site specific pseudouridine detection can provide to the understanding of the intrinsic ribosomal changes occurring in human tumors.
... Such uridines are in positions 1911, 1915, and 1917 of 23S rRNA, in a region known as helix-loop 69 (HL69) (Leppik et al. 2007). This structure constitutes a bridge between ribosomal subunits and ensures translation fidelity (Jiang et al. 2014), which makes it very important for translation to take place. It has been shown that, at least in vitro, mutations at Ψ1917 inhibit translation (Liiv et al. 2005), and this could represent a strongly deleterious effect in vivo Ofengand et al. 2001). ...
The isomerases are a unique enzymatic class of enzymes that carry out a great diversity of chemical reactions at the intramolecular level. This class comprises about 300 members, most of which are involved in carbohydrate and terpenoid/polyketide metabolism. Along with oxidoreductases and translocases, isomerases are one of the classes with the highest ratio of paralogous enzymes. Due to its relatively small number of members, it is plausible to explore it in greater detail to identify specific cases of gene duplication. Here, we present an analysis at the level of individual isomerases and identify different members that seem to be involved in duplication events in prokaryotes. As was suggested in a previous study, there is no homogeneous distribution of paralogs, but rather they accumulate into a few subcategories, some of which differ between Archaea and Bacteria. As expected, the metabolic processes with more paralogous isomerases have to do with carbohydrate metabolism but also with RNA modification (a particular case involving an rRNA-modifying isomerase is thoroughly discussed and analyzed in detail). Overall, our findings suggest that the most common fate for paralogous enzymes is the retention of the original enzymatic function, either associated with a dosage effect or with differential expression in response to changing environments, followed by subfunctionalization and, to a much lesser degree, neofunctionalization, which is consistent with what has been reported elsewhere.
... Structural and comparative studies have revealed that the first and the last nucleobase of the anticodon loop (bases 32 and 38) tend to form a non-Watson-Crick pair with a single hydrogen bond [22]. This interaction can be facilitated by a pseudouridine, as it forms more stable base pairs and stacking interactions compared to the unmodified uridine [23][24][25]. Interaction between bases 32 and 38 is transiently lost during +1 frameshifting [26,27]. Thus, pseudouridine at position 38/39 (isomerized by TruA) or 32 (isomerized by RluA) could help to keep codon reading frame by local stabilization of anticodon loop structure. ...
Delicate variances in the translational machinery affect how efficiently different organisms approach protein synthesis. Determining the scale of this effect, however, requires knowledge on the differences of mistranslation levels. Here, we used a dual-luciferase reporter assay cloned into a broad host range plasmid to reveal the translational fidelity profiles of Pseudomonas putida, Pseudomonas aeruginosa and Escherichia coli. We observed that these profiles are surprisingly different, whereas species more prone to translational frameshifting are not necessarily more prone to stop codon readthrough. As tRNA modifications are among the factors that have been implicated to affect translation accuracy, we also show that translational fidelity is context-specifically influenced by pseudouridines in the anticodon stem-loop of tRNA, but the effect is not uniform between species.
... Expanded contour plots of the NOESY spectra corresponding to the interactions between the base H6/H8 and H1′/H5 protons with sequential pathways traced for both duplexes are shown in Fig. 2. For the Ψ residues at position 5 (Ψ5), characteristic upfield shifts of the H1′ resonances relative to the remaining H1′ resonances were observed (Supplementary Tables S1, S3). This tendency is a signature of Ψ modification and is consistent with previous NMR reports regarding pseudouridine-containing RNAs 15,23,33 . Similar to 1 H, the 13 C chemical shifts of C1′-Ψ5 differ significantly from the ranges observed for the other anomeric carbon resonances (Supplementary Tables S2, S4). ...
Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modiication on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ afected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.
... More specifically, Ψ can stabilize RNA 3D conformation by water bridge formation, as demonstrated in tRNAs 7,8 . A cluster of Ψs can modulate the conformation of a large RNA domain as shown for helix 69 -H69 -in rRNA 9 . It is now firmly established that some evolutionarily conserved Ψs in tRNAs and rRNAs are required for accurate function of these non-coding RNAs (ncRNAs) in translation (for review 2 , and [10][11][12][13][14][15][16][17][18][19][20]. ...
Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10
and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure
the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs).
Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the
hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution
of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent
activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes
are viable, but display a thermosensitive slow growth phenotype. We also generated a
single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in
vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl
transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for
rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and
in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore,
our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for
which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent
activity of Cbf5.
... Pseudouridines are not only found in rRNA but in all types of cellular RNA including mRNAs, tRNAs, small nuclear RNAs, small nucleolar RNAs and long-noncoding RNAs [11][12][13]. In general, pseudouridines are thought to play a role in RNA stabilization, structure and correct RNA folding [14][15][16], but the function of pseudouridine is not yet fully understood. Interestingly, this modification can be induced in small nuclear RNAs by stress conditions [17,18], and the presence of pseudouridine in eukaryote mRNA may suggest a role in gene expression regulation [19,20]. ...
The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure-function analysis to determine the structural elements both in RluE as well as in 23S rRNA required for RNA-protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 as well as the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome biogenesis. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome formation.
... Pseudouridine base pairs with any of the four major bases are more stable than their U equivalents (53,54). Three pseudouridine residues in the H69 stabilize both base-pairing and base stacking in this functionally important structural element (55). High-resolution crystal structure of the E. coli ribosome demonstrates that six of the seven well-ordered pseudouridines are involved in watermediated contacts between the pseudouridine N1 imino groups in the major groove and the rRNA phosphate backbone, which can explain the stabilizing effect of pseudouridine (56). ...
Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly.
