Figure 3 - uploaded by Christiane Branlant
Content may be subject to copyright.
The aCBF5-aNOP10-aGAR1 complex can efficiently modify a tRNA lacking a 3 0 CCA sequence. Time-course experiments of É55 formation in the variant tRNA Asp-ÁCCA by the E. coli TruB enzyme or various combinations of the aCBF5 (C), aNOP10 (N) and aGAR1 (G) proteins (same conditions as in Figure 2).
Source publication
Protein aNOP10 has an essential scaffolding function in H/ACA sRNPs and its interaction with the pseudouridine(Ψ)-synthase aCBF5 is required for the RNA-guided RNA:Ψ-synthase activity. Recently, aCBF5 was shown to catalyze the isomerization of U55 in tRNAs without the help of a guide sRNA.
Here we show that the stable anchoring of aCBF5 to tRNAs re...
Similar publications
The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His6-tag. The resulting recombinant protei...
Citations
... Ψ55 is commonly found in eubacteria, archaea, and eukaryotes [29,30]. The archaeal Ψ55 synthases are Cbf5 and Pus10 [31][32][33][34]. Since the archaeal Pus10 can convert U54 and U55 to Ψ modifications, Pus10 is a multiple-site-specific pseudouridine synthase. ...
Eukaryotic precursor tRNAs (pre-tRNAs) often have an intron between positions 37 and 38 of the anticodon loop. However, atypical introns are found in some eukaryotes and archaea. In an early-diverged red alga Cyanidioschyzon merolae, the tRNAIle(UAU) gene contains three intron coding regions, located in the D-, anticodon, and T-arms. In this study, we focused on the relationship between the intron removal and formation of pseudouridine (Ψ), one of the most universally modified nucleosides. It had been reported that yeast Pus1 is a multiple-site-specific enzyme that synthesizes Ψ34 and Ψ36 in tRNAIle(UAU) in an intron-dependent manner. Unexpectedly, our biochemical experiments showed that the C. merolae ortholog of Pus1 pseudouridylated an intronless tRNAIle(UAU) and that the modification position was determined to be 55 which is the target of Pus4 but not Pus1 in yeast. Furthermore, unlike yeast Pus1, cmPus1 mediates Ψ modification at positions 34, 36, and/or 55 only in some specific intron-containing pre-tRNAIle(UAU) variants. cmPus4 was confirmed to be a single-site-specific enzyme that only converts U55 to Ψ, in a similar manner to yeast Pus4. cmPus4 did not catalyze the pseudouridine formation in pre-tRNAs containing an intron in the T-arm.
... These conditions also impact on patients quality life (Fernandes & Caliri, 2000;Blanes et al., 2004). In front of that scenario, biomodulator therapies are able to modulate tissue repair because of their anti-inflammatory and analgesic effects, besides cellular and molecular signaling (Muller et al., 2007). ...
Tissue repair process consists in a complex and dynamic event straightly linked to tissues self-regeneration capability. In that context, it may also occur total or partial replacement of the original tissues through an extracellular matrix with significant collagen and elastin expression that results in fibroplasia. Aiming to optimizing the repair, it has been used some resources able to biomodulate the action of different kinds of tissue cells such as laser photobiomodulation (LP), light emitting diodes (LEDs), ozone therapy and plasma jet. This narrative literature review aims to describe the different phases of tissue repair and discuss about biomodulator therapies commonly applied to stimulate that process. Manuscripts discussing the proposal theme, which have been fully available were selected from Pubmed, Embase, Cochrane and Scielo database. The manuscripts must be written in English or Portuguese language, according to the exclusion and inclusion criteria designed for that study. Sixty manuscripts were selected. It may be noticed that biomodulator therapies have been used in health areas. However, studies evaluating the influence of plasma jet on tissue repair are still scarce. Preliminary results from such studies suggest that plasma jet therapy may perform important antiinflammatory, analgesic and biostimulating effects.
... In Archaea, Cbf5 can function as a stand-alone Ψ-synthase for tRNA Ψ55 formation, at least in vitro [81][82][83][84]. This guide-independent tRNA specific activity of archaeal Cbf5 is enhanced in vitro by the proteins Nop10 and Gar1, probably by stabilizing the active conformation of Cbf5 [82,[85][86][87]. It is worth mentioning that the Haloferax volcanii cbf5 deletion strain still contains tRNA carrying Ψ55, attesting that Cbf5 is not the main physiological Ψ55 synthase, at least in this archaeon [88]. ...
The high conservation of nucleotides of the T-loop, including their chemical identity, are hallmarks of tRNAs from organisms belonging to the three Domains of Life. These structural characteristics allow the T-loop to adopt a peculiar intraloop conformation able to interact specifically with other conserved residues of the D-loop, which ultimately folds the mature tRNA in a unique functional canonical L-shaped architecture. Paradoxically, despite the high conservation of modified nucleotides in the T-loop, enzymes catalyzing their formation depend mostly on the considered organism, attesting for an independent but convergent evolution of the post-transcriptional modification processes. The driving force behind this is the preservation of a native conformation of the tRNA elbow that underlies the various interactions of tRNA molecules with different cellular components.
... The ⌿55 modification in tRNA Trp is likely to be performed by archaeal Pus10 (93-95) or archaeal Cbf5 (95)(96)(97)(98). Because Sulfolobus solfaraticus Pus7 has been reported to possess weak activity for ⌿13 formation (99), the homologous protein (annotated as TruD [100] in the database [54]; Tk2302 gene product) may form ⌿13 in tRNA Trp from T. kodakarensis. ...
Thermococcus kodakarensis is a hyperthermophilic archaeon that can grow at 60 to 100°C. The sequence of tRNA Trp from this archaeon was determined by liquid chromatography/mass spectrometry. Fifteen types of modified nucleoside were observed at 21 positions, including 5 modifications at novel positions; in addition, methylwyosine at position 37 was newly observed in an archaeal tRNA Trp . The construction of trm11 (Δ trm11 ) and other gene disruptant strains confirmed the enzymes responsible for modifications in this tRNA. The lack of 2-methylguanosine (m ² G) at position 67 in the trm11 trm14 double disruptant strain suggested that this position is methylated by Trm14, which was previously identified as an m ² G6 methyltransferase. The Δ trm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures.
... Archaea and Eukarya contain Cbf5, a box H/ACA guide RNA-dependent Ψ synthase, which structurally belongs to the TruB family of Ψ synthases (Koonin 1996;Watanabe and Gray 2000;Mueller and Ferre-D'Amare 2009). In vitro, archaeal Cbf5 can also produce Ψ55 in tRNA and Ψ at some positions in rRNA in a guide RNA-independent manner (Roovers et al. 2006;Gurha et al. 2007;Muller et al. 2007Muller et al. , 2008; Kamalampeta and Kothe 2012), but this activity of Cbf5 has only been shown in vivo for rRNA (Fujikane et al. 2018). On the other hand, archaeal Pus10 (PsuX), a Ψ synthase distinct from the TruB family (Watanabe and Gray 2000;Mueller and Ferre-D'Amare 2009;Rintala-Dempsey and Kothe 2017), can produce both Ψ54 and Ψ55 in tRNA, both in vitro and in vivo (Gurha and Gupta 2008;Joardar et al. 2013). ...
The nearly conserved U54 of tRNA is mostly converted to a version of ribothymidine (T) in Bacteria and eukaryotes and to a version of pseudouridine (Ψ) in Archaea. Conserved U55 is nearly always modified to Ψ55 in all organisms. Orthologs of TrmA and TruB that produce T54 and Ψ55, respectively, in Bacteria and eukaryotes are absent in Archaea. Pus10 produces both Ψ54 and Ψ55 in Archaea. Pus10 orthologs are found in nearly all sequenced archaeal and most eukaryal genomes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in most archaeal tRNAs and some animal tRNAs, but its absence from yeast and bacteria. Moreover, Ψ54 is found in several tRNAs that function as primers for retroviral DNA synthesis. Previously, no eukaryotic tRNA Ψ54 synthase had been identified. We show here that human Pus10 can produce Ψ54 in select tRNAs, including tRNALys3, the primer for HIV reverse transcriptase. This synthase activity of Pus10 is restricted to the cytoplasm and is distinct from nuclear Pus10, which is known to be involved in apoptosis. The sequence GUUCAm1AAUC (m1A is 1-methyladenosine) at position 53-61 of tRNA along with a stable acceptor stem results in maximum Ψ54 synthase activity. This recognition sequence is unique for a Ψ synthase in that it contains another modification. In addition to Ψ54, SF9 cells-derived recombinant human Pus10 can also generate Ψ55, even in tRNAs that do not contain the Ψ54 synthase recognition sequence. This activity may be redundant with that of TruB. © 2019 Deogharia et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
... For instance, Cbf5, which is the only representative of the TruB family in archaea and acts as the catalytic subunit of the H/ACA sRNPs 30,31 , may also function as a stand-alone enzyme. In vitro assays using the P. abyssi Cbf5 showed that it is able to catalyze Ψ 55 formation in the loop of the TΨC arm of elongator tRNAs 26,[44][45][46][47][48][49] . One argument against this hypothesis is the finding that Cbf5 is not essential for the generation of this modification in vivo in Haloferax volcanii, in which this activity is carried out by the Pus10 PUS 26,[50][51][52][53][54] . ...
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.
... In addition to Cbf5 and L7Ae, archaeal H/ACA RNP also contains Nop10p and Gar1 proteins (for review, see Yip et al. 2013). Interestingly, the archaeal Cbf5 pseudouridine synthase can also perform uridine isomerization in an RNA-independent, or standalone, mechanism on ACA-less-tRNA substrates at position U55, and U2603 of 23S rRNA (Roovers et al. 2006;Muller et al. 2007Muller et al. , 2008Zhou et al. 2011). Furthermore, Cbf5 has been shown to preferentially bind the H/ACA box RNA over its stand-alone substrate, resulting in similar inhibition of the stand-alone activity (Zhou et al. 2011). ...
Archaeal fibrillarin (aFib) is a well-characterized S-Adenosyl methionine (SAM)-dependent RNA 2'-O-methyltransferase that is known to act in a large C/D ribonucleoprotein (RNP) complex together with Nop5 and L7Ae proteins and a box C/D guide RNA. In the reaction, the guide RNA is critical to direct the methylation reaction to a specific site in tRNA or rRNA by sequence complementarity. Here we show that Pyrococcus abyssi aFib-Nop5 heterodimer can alone perform a SAM-dependent 2'-O-methylation of 16S and 23S ribosomal RNAs in vitro independently of L7Ae and C/D guide RNAs. Using tritium-labeling, mass spectrometry and reverse transcription analysis, we identified three in vitro 2'-O-methylated positions in the 16S rRNA of P. abyssi, positions lying outside of previously reported pyrococcal C/D RNP methylation sites. This newly discovered stand-alone activity of aFib-Nop5 may provide an example of an ancestral activity retained in enzymes that were recruited to larger complexes during evolution.
... Bacterial TruB (and Pus4, its eukaryal ortholog) produces Ψ55 in tRNAs in a guide RNA-independent manner. Cbf5 has also been shown in vitro to produce Ψ55 in tRNAs and some other Ψ in rRNAs in a guide RNA-independent manner and this activity is enhanced by Gar1 and Nop10 (Roovers et al. 2006;Gurha et al. 2007;Muller et al. 2007Muller et al. , 2008; Kamalampeta and Kothe 2012). Based on the structural and functional similarities of Cbf5 and TruB, certain common features about their mechanisms of RNA recognition and action have been proposed. ...
... Archaeal Cbf5 in vitro has been shown to have both RNAguided and guide RNA-independent pseudouridylation activities (Baker et al. 2005;Charpentier et al. 2005;Roovers et al. 2006;Gurha et al. 2007;Muller et al. 2007Muller et al. , 2008. ...
In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNA-guided pseudouridylation activity.
... Archaeal protein Cbf5 (alias aCBF5) has related features with the E. coli TruB enzyme as it is able to generate in vitro the universally conserved Ψ 55 in elongator tRNAs (Fig. 1A 1 ; Roovers et al. 2006;Gurha et al. 2007;Muller et al. 2007;Kamalampeta and Kothe 2012). A pseudouridine can also be introduced at this position by the archaeal Pus10 enzyme in vitro and in vivo (Roovers et al. 2006;Gurha and Gupta 2008;Blaby et al. 2011;Chatterjee et al. 2012;Joardar et al. 2013;Kamalampeta et al. 2013). ...
... In agreement with the feature of the bacterial TruB enzyme, the histidine equivalent to H43 at position 77 (H77) in archaeal Cbf5 is also essential for U 55 tRNA modification in vitro, but does not strongly impact the activity of H/ ACA sRNP (Muller et al. 2007). Based on the crystallographic structure of the substrate-bound state of the H/ACA sRNP, Duan et al. (2009) proposed that histidine H60, which is conserved in Cbf5 orthologs but not in TruB orthologs, would substitute H77 and have a functional role in U extrusion into the active site of Cbf5 (Fig. 1B 2 ). ...
... These data revealed that although the substitution of H60 or H77 did not fully impair substrate modification, substitution of H77 led to a slower enzyme than H60. Activity of variant H77A appeared even slower than in our previous measurements (Muller et al. 2007). The H60A/H77A variant sRNP was inactive, which was in agreement with the low amounts of CII and CII ′ complexes. ...
In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)-the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U55 in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.
© 2015 Tillault et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
... We previously used the Pab91 RNP enzyme from the hyperthermophilic archaeon P. abyssi as a model for structure-function analyses of H/ACA RNPs [17,37,39]. In vitro activity of the enzyme was measured on the 22-mer RNA substrate 22eU (Fig. 1A) corresponding to a fragment of the P. abyssi 23S rRNA encompassing residue U2685 targeted for pseudouridylation by the Pab91 H/ACA sRNA [12]. ...
The box H/ACA small ribonucleoprotein particles (H/ACA sRNPs) are RNP enzymes that isomerize uridines (U) into pseudouridines (Ψ) in archaeal RNAs. The RNA component acts as a guide by forming base-pair interactions with the substrate RNA to specify the target nucleotide of the modification to the catalytic subunit Cbf5. Here, we have analyzed association of an H/ACA sRNP enzyme from the hyperthermophilic archaeon Pyrococcus abyssi with synthetic substrate RNAs of different length and with target nucleotide variants, and estimated their turnover at high temperature. In these conditions, we found that a short substrate, which length is restricted to the interaction with RNA guide sequence, has higher turnover rate. However, the longer substrate with additional 5' and 3' sequences non-complementary to the guide RNA is better discriminated by the U to Ψ conversion allowing the RNP enzyme to distinguish the modified product from the substrate. In addition, we identified that the conserved residue Y179 in the catalytic center of Cbf5 is crucial for substrate selectivity.
Copyright © 2015. Published by Elsevier B.V.
