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Predicted and experimentally identified target sites of the identified P. abyssi H/ACA sRNAs and comparison with P. furiosus
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How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational
search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified
17 Ψ residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA...
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... electro- transfer on a Hybond-N+ membrane (Amersham) and by UV-crosslinking, a pre-hybridization was carried out for 1 h at 588C in SSPE buffer (0.9 M NaCl, 47 mM Na 2 HPO 4 -2H 2 O, 6 mM EDTA pH 7.4, containing 1 g/l Ficoll, 1 g/l polyvinylpyrolidone, 1 g/l BSA, 0.5% SDS). Oligonucleotide probes complementary to the predicted H/ACA sRNAs (Table S1 in Supplementary data) were 5 0 -end labelled with [g-32 P]ATP and T4 polynucleotide kinase for 1 h at 378C. Hybridization was carried out at 588C for 16 h in the presence of 100 ng of the labelled oligonucleotide. ...
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... of CMCT modifications were identified by primer extension analysis, using the AMV RT (QBiogene, USA). The sequences of the 5 0 -end labelled primers that we used are given in Table S1 (Supplementary data). RNA sequencing was done on 20 mg of P. abyssi total RNA. ...
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... Pab19, Pab21, Pab35, Pab40, Pab91, Pab105 and Pab160 sRNA sequences were PCR amplified from the P. abyssi GE5 genomic DNA using a forward primer containing the T7 RNA polymerase promoter and a second primer complementary to the expected 3 0 end of the sRNA (Table S1 in Supplementary data). The amplified DNA fragments were cloned in the pTAdv vector (Clontech) and sequenced. ...
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... together, 14 and 7 target sites were predicted in 23S and 16S rRNAs, respectively. Note that motif 1 in sRNA Pab35 and sRNA Pab160 are both predicted to guide modifications at position 922 in 16S rRNA and at position 2672 in 23S rRNA (Table 1). Only 8 of the 17 sites that we predicted for the five previously identified P. furiosus H/ACA sRNAs had been proposed (22). ...
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... this end, total RNA from P. abyssi was treated with CMCT with or without further alkaline incubation. Positions of the alkaline-resistant É residues were detected by primer-extension analyses using a large series of oligonucleotide primers (Table S1 in Supplementary data). Altogether, the 16S and 23S rRNA segments that were probed represented 20 and 27% of the entire molecules, respectively. ...
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... experimental analysis confirmed É formation at 12 of the 21 predicted sites (positions 27, 891, 922 in 16S rRNA, and positions 1932, 2549, 2554, 2588, 2672, 2685, 2697, 2794and 2930 (Table 1 and Figure 5). In addition, the absence of É formation was clearly demonstrated at six of the predicted positions (positions 892, 995 and 1122 in 16S rRNA and 278, 2250, and 2552) ( Table 1 and Figure 5). ...
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... experimental analysis confirmed É formation at 12 of the 21 predicted sites (positions 27, 891, 922 in 16S rRNA, and positions 1932, 2549, 2554, 2588, 2672, 2685, 2697, 2794and 2930 (Table 1 and Figure 5). In addition, the absence of É formation was clearly demonstrated at six of the predicted positions (positions 892, 995 and 1122 in 16S rRNA and 278, 2250, and 2552) ( Table 1 and Figure 5). Note that three of these unmodi- fied positions were previously proposed to be modified in P. furiosus (22) . ...
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... expression of each gene encoding a putative H/ACA sRNA was tested by Northern blot analysis of P. abyssi total RNA extracts, as described in Materials and Methods (Lane 1 in each panel). The nucleotide sequences of the specific 5 0 -end radio-labelled probes used for each sRNA are given in Table S1 in Supplementary data. A 5 0 -end labelled DNA ladder was loaded in Lane 2. The detected transcripts are shown with an arrow on the left of each autoradiogram. ...
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... the RNAs were extracted, digested, and fractionated by thin layer chromatography (as described in Materials and Methods) ( Figure 6). Only 15 of the 21 predicted sites were found to be modified by the reconstituted sRNPs and the rates of U to É conversions were ranging from 30 to 100% ( Figure 6 and Table 1). The rRNA segments predicted to be modified by two distinct H/ACA motifs were in fact modified by only one of them (motif 1 in sRNA Pab35 modifies position 2672 in 23S rRNA and sRNA Pab160 acts at position 922 in 16S rRNA). ...
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... we found that all the H/ACA sRNAs that contain a single H/ACA motif (Pab21, Pab91, Pab160 and Pab19) guide modification at a unique position in rRNAs. Altogether, the 11 H/ACA motifs of the 7 P. abyssi sRNAs can guide U to É conversion at 15 sites in the P. abyssi rRNAs ( Table 1). Presence of É residue was detected at 12 of these sites ( Figure 5 and Table 1). ...
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... the 11 H/ACA motifs of the 7 P. abyssi sRNAs can guide U to É conversion at 15 sites in the P. abyssi rRNAs ( Table 1). Presence of É residue was detected at 12 of these sites ( Figure 5 and Table 1). As mentioned above, for technical reasons, modification could not be tested experimentally at the three other positions. ...
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... we detected no guide H/ACA sRNA for residues É2585 and É2603 in P. abyssi 23S rRNA, and as the aCBF5/ aNOP10/aGAR1 complex can modify position 55 in tRNAs in the absence of guide sRNA (50-52), we tested the in vitro activity of this complex at these two 23S rRNA positions. The assays were performed using two 20-nt long rRNA fragments (Table S1) containing, respectively, residue U2585 or U2603 (Figure 7). The aCBF5/ aNOP10/aGAR1 complex was active on the rRNA frag- ment containing residue U2603 (46% yield) (Figure 7), but not on that containing residue U2585. ...
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... that two other proposed target sites in P. furiosus rRNAs (corresponding to positions 892 in 16S rRNA and 2552 in P. abyssi 23S rRNA and which were expected to be guided by sRNAs Pf1 and Pf3 respectively, Table 1), are invalidated by our experimental rules. In addition, some bona fide target sites were not previously predicted in P. furiosus rRNAs (Table 1). Altogether, these data strengthen the importance to verify experimentally the proposed H/ACA target sites. ...
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Citations
... sR136 guides the modification of U1009 in the 16S rRNA (Figure 4 D), as confirmed through in vitro modification assays (Figure 6 E). Interestingly, the equivalent site U1017 in P. abyssi is also pseudouridylated, but it uses a classic H / ACA RNA as guide ( 23 ). Unlike other H / ACA RNAs that typically have two guide sequences, sR148 contains a single 3 guide sequence that was predicted to target two closely located sites, U2060 and U2066, in the 23S rRNA (Figure 4 E). ...
... Previous studies have identified a homolog of sR435 in S. solfataricus , but only a single target equivalent to 23S-U2597 was predicted ( 22 ). On the other hand, a large fraction of predicted targets of P. abyssi H / ACA RNAs were not confirmed ( 23 ). ...
Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.
... Hence, RNA-guided modification is not the prerogative of snoRNPs, and It is probable that eukaryotic snoRNAs originate from an ancestor present before the separation between Eukarya and Archaea (for a review, see [34]). Indeed, small RNAs with box C/D or box H/ACA snoRNA features, named sRNAs, are also acting for RNA-guided modification in archaeons [35][36][37][38][39][40][41] (for a review, see [42,43]). This phylogenetic feature highlights the essentiality of the cellular activity of these systems that could have expanded in eukaryotic cells with other functions and mechanisms of action. ...
Box C/D small nucleolar RNAs (C/D snoRNAs) represent an ancient family of small non-coding RNAs that are classically viewed as housekeeping guides for the 2′-O-methylation of ribosomal RNA in Archaea and Eukaryotes. However, an extensive set of studies now argues that they are involved in mechanisms that go well beyond this function. Here, we present these pieces of evidence in light of the current comprehension of the molecular mechanisms that control C/D snoRNA expression and function. From this inventory emerges that an accurate description of these activities at a molecular level is required to let the snoRNA field enter in a second age of maturity.
... To mediate further processing in yeast, H/ACA sRNAs are polyadenylated by the poly(A) polymerase Pap1 or the alternative Tfr4, bound by polyA binding protein (Pab2 in fission yeast) and subsequently processed by the nuclear exosome (van Hoof et al., 2000;Grzechnik and Kufel, 2008;Lemay et al., 2010;Berndt et al., 2012). As there are only few pseudouridines in archaeal rRNA and thus only few H/ACA RNAs, the transcription and maturation of archaeal H/ACA RNAs has not been studied in detail, but many archaeal H/ACA RNAs have been identified (Rozhdestvensky et al., 2003;Muller et al., 2007Muller et al., , 2008Randau, 2015;Toffano-Nioche et al., 2015;Clouet-d'Orval et al., 2018). ...
During ribosome synthesis, ribosomal RNA is modified through the formation of many pseudouridines and methylations which contribute to ribosome function across all domains of life. In archaea and eukaryotes, pseudouridylation of rRNA is catalyzed by H/ACA small ribonucleoproteins (sRNPs) utilizing different H/ACA guide RNAs to identify target uridines for modification. H/ACA sRNPs are conserved in archaea and eukaryotes, as they share a common general architecture and function, but there are also several notable differences between archaeal and eukaryotic H/ACA sRNPs. Due to the higher protein stability in archaea, we have more information on the structure of archaeal H/ACA sRNPs compared to eukaryotic counterparts. However, based on the long history of yeast genetic and other cellular studies, the biological role of H/ACA sRNPs during ribosome biogenesis is better understood in eukaryotes than archaea. Therefore, this review provides an overview of the current knowledge on H/ACA sRNPs from archaea, in particular their structure and function, and relates it to our understanding of the roles of eukaryotic H/ACA sRNP during eukaryotic ribosome synthesis and beyond. Based on this comparison of our current insights into archaeal and eukaryotic H/ACA sRNPs, we discuss what role archaeal H/ACA sRNPs may play in the formation of ribosomes.
... Furthermore, binding of Cbf5 to the ACA box is essential for the formation of correct 3 ′ terminus of the guide RNA, probably by preventing the action of exonucleases, especially for the RNAs derived from introns. Archaeal box H/ACA RNAs generally have one to three stem-loops or H/ACA motifs (Tang et al. 2002;Muller et al. 2008) with no H box in one stem-loop H/ACA RNA. Archaeal H/ACA motif (see Fig. 1A) contains a proximal or lower (P1) and a distal or upper (P2) stem separated by an internal loop called a "pseudouridylation pocket" (Ψ pocket). ...
... This spacing includes only the P1b side of the P1 stem and the section of the PS2 side of the Ψ pocket up to the last base that pairs with the target RNA. The P2 stem in Archaea has either a K-turn or its variant, a K-loop (Rozhdestvensky et al. 2003;Hamma and Ferré-D'Amaré 2004;Muller et al. 2008), both of which contain two tandem sheared G:A pairs. Generally, the distance from the distal G:A pair to the base of the P2 stem is 9-10 bases (or base pairs) (Li and Ye 2006). ...
... We observed some Ψ formation in vitro, when pairing of the target RNA is only retained with the 3 ′ side of the Ψ pocket, but not when it is retained only on the 5 ′ side (M5Gi vs. M3Gi in row IV, Fig 7A, and SL2-M5Gi vs. SL2-M3Gi in row II, Fig. 7B). The greater importance of 3 ′ pairing than 5 ′ pairing was also observed by others (Muller et al. 2008). However, the pairing of target RNA with both sides of the Ψ pocket is required in vivo, probably to provide additional stability. ...
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 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 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 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 Cbf5 protein affect pseudouridylation by the two stem-loops of sR-h45 differently. This study will help elucidate the conditions for production of non-constitutive Ψ's, determine functions for orphan H/ACA RNAs and in target designing.
... 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.
... Furthermore, Cbf5 may have a stand-alone activity in modification of rRNA. Indeed, despite of a deep computer analysis, we failed to identify any H/ACA sRNA able to guide formation of the two Ψs respectively detected at positions 2585 and 2603 in the P. abyssi in 23S rRNA 55 , and we found that recombinant Cbf5 in presence of proteins Nop10 and Gar1 can modify in vitro residue U 2603 in a transcribed 23S rRNA fragment that can fold into a stem-loop structure showing some similarity with the TΨC arm of tRNAs 48,55 . As this TΨC-like-loop structure has not been predicted in classical 23S rRNA folding models, the possible RNA-independent activity of Cbf5 in archaeal 23S rRNA modification still remains to be demonstrated in vivo. ...
... Furthermore, Cbf5 may have a stand-alone activity in modification of rRNA. Indeed, despite of a deep computer analysis, we failed to identify any H/ACA sRNA able to guide formation of the two Ψs respectively detected at positions 2585 and 2603 in the P. abyssi in 23S rRNA 55 , and we found that recombinant Cbf5 in presence of proteins Nop10 and Gar1 can modify in vitro residue U 2603 in a transcribed 23S rRNA fragment that can fold into a stem-loop structure showing some similarity with the TΨC arm of tRNAs 48,55 . As this TΨC-like-loop structure has not been predicted in classical 23S rRNA folding models, the possible RNA-independent activity of Cbf5 in archaeal 23S rRNA modification still remains to be demonstrated in vivo. ...
... As this TΨC-like-loop structure has not been predicted in classical 23S rRNA folding models, the possible RNA-independent activity of Cbf5 in archaeal 23S rRNA modification still remains to be demonstrated in vivo. Of note, the previous data did not reveal any possible activity of either Cbf5 alone or of the Cbf5-Nop10-Gar1 protein complex at position U 2585 , raising the question of the catalyst used at this position 55 . ...
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.
... Each archaeal H/ACA stem-loop structure contains Kturn or K-loop structural motifs which binds L7Ae. The complete archaeal H/ACA sRNPs contain three other proteins: the pseudouridine synthetase aCbf5 and aNop10 and aGar1 required to reinforce aCBF5 activity (Charpentier, Muller and Branlant 2005;Muller et al. 2008). The RNA component of the complex serves as a guide that base pairs with the RNA substrate and directs the enzyme (aCbf5 or aNop1) to carry out the pseudouridine formation or methyl transfer at a specific site. ...
... Interestingly, in Archaea, the RNA-dependent reactions involve more than 100 small box guide RNAs that target specific sites of rRNAs (Muller et al. 2008). In addition, these small-guide RNAs have been suggested to serve as 'RNA chaperones' in facilitating rRNA folding (Bachellerie et al. 1995;Steitz and Tycowski 1995;Dennis et al. 2015). ...
RNA processing pathways are at the center of regulation of gene expression. All RNA transcripts undergo multiple maturation steps in addition to covalent chemical modifications to become functional in the cell. This includes destroying unnecessary or defective cellular RNAs. In Archaea, information on mechanisms by which RNA species reach their mature forms and associated RNA-modifying enzymes is still fragmentary. To date, most archaeal actors and pathways have been proposed in light of information gathered from Bacteria and Eukarya. In this context, this review provides a state of the art overview of archaeal endoribonucleases and exoribonucleases that cleave and trim RNA species and also of the key small archaeal proteins that bind RNAs. Furthermore, synthetic up-to-date views of processing and biogenesis pathways of archaeal transfer and ribosomal RNAs as well as of maturation of stable small non-coding RNAs such as CRISPR RNAs, small C/D and H/ACA box guide RNAs, and other emerging classes of small RNAs are described. Finally prospective post-transcriptional mechanisms to control archaeal messenger RNA quality and quantity are discussed.
... Generally, that ORF corresponds to the sRNA target which presents complete complementarity to the cis-encoded sRNA. In addition, there have also been other types of RNA identified in Archaea: small nucleolar RNAs (snoRNAs) involved in the modifications of ribosomal RNA, whose presence was originally believed to be restricted to eukaryotic organisms [10]; sRNAs involved in the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas prokaryotes immune system called crRNAs [11]; and sRNAs that derive from transfer RNAs called tRFs [12]. ...
... The application of bioinformatic approaches and high-throughput sequencing systems for the analysis of complementary DNA (cDNA) libraries, RNA-Sequencing (RNA-Seq), have made it easier to understand the transcriptome and discover new sRNAs. However, to date, the identification of sRNAs using RNA-Seq analysis has only been carried out on seven species of Archaea under specific conditions: Archaeoglobus fulgidus [15], Sulfolobus solfataricus [16], Pyrococcus abyssi [10], Methanosarcina mazei [17], Haloferax volcanii [13], Pyrobaculum sp. [18], and Thermococcus kodakaraensis [19]. ...
Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in the domain Archaea and their possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea,Haloferax mediterraneihas been used as a model microorganism. The bioinformatic approach has allowed for the prediction of 295 putative sRNAs genes in the genome ofH. mediterranei, 88 of which have been verified by means of RNA-Sequencing (RNA-Seq). The secondary structure of these sRNAs and their possible targets have been identified. Curiously, some of them present as possible target genes relating to nitrogen assimilation, such as glutamate dehydrogenase and the nitrogen regulatory PII protein. Analysis of RNA-Seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and bioinformatic approaches used in this work have allowed for the identification of new sRNAs inH. mediterranei, some of which show different expression patterns depending on the nitrogen source. This suggests that these sRNAs could be involved in the regulation of nitrogen assimilation and can constitute an important gene regulatory network.
... Importantly, several critical differences are evident between archaeal and eukaryotic H/ACA RNPs. First, archaea utilize a wide variety of H/ACA guide RNAs containing one to three hairpins (23,24) whereas most eukaryotes have strictly conserved guide RNAs with two hairpins (25)(26)(27). Second, archaeal guide RNAs contain a kink-turn or kink-loop motif which is specifically recognized with low nanomolar affinity by the protein L7Ae (28). ...
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.
... 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.
