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. 2011 Sep 1;8(5):782–791. doi: 10.4161/rna.8.5.16015

2′-O-methylation of the wobble residue of elongator pre-tRNAMet in Haloferax volcanii is guided by a box C/D RNA containing unique features

Archi Joardar 1, Srinivas R Malliahgari 1, Geena Skariah 1, Ramesh Gupta 1,✉
PMCID: PMC3256356  PMID: 21654217

Abstract

The wobble residue C34 of Haloferax volcanii elongator tRNAMet is 2′-O-methylated. Neither a protein enzyme nor a guide RNA for this modification has been described. In this study, we show that this methylation is guided by a box C/D RNA targeting the intron-containing precursor of the tRNA. This guide RNA is starkly different from its homologs. This unique RNA of approximately 75 bases, named sR-tMet, is encoded in the genomes of H. volcanii and several other haloarchaea. A unique feature of sR-tMet is that the mature RNA in H. volcanii is substantially larger than its predicted size, whereas those in other haloarchaea are as predicted. While the 5′-ends of all tested haloarchaeal sR-tMets are equivalent, H. volcanii sR-tMet possesses an additional 51-base extension at its 3′ end. This extension is present in the precursor but not in the mature sR-tMet of Halobacterium sp, suggesting differential 3′-end processing of sR-tMet in these two closely related organisms. Archaeal box C/D RNAs mostly contain a K-loop at the C′/D′ motif. Another unique feature of sR-tMet is that its C′/D′ motif lacks either a conventional K-turn or a K-loop. Instead, it contains two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in the non-canonical stem; the latter may form an alternative K-turn. Gel shift assays indicate that the L7Ae protein can form a stable complex with this unusual C′/D′ motif, suggesting a novel RNA structure for L7Ae interaction.

Key words: sRNA, snoRNA, Guide RNA, tRNA modification, RNA 2′-O-methylation, RNA-guided modification, Ribonucleoprotein, Box C/D RNA

Introduction

Eukaryal nucleoli contain small nucleolar RNAs (snoRNAs) that are involved in processing of pre-rRNAs and other RNAs.1–10 These snoRNAs function by associating with proteins to form ribonulceoprotein complexes (snoRNPs). One function of these snoRNAs is to guide modification of specific nucleotides by base pairing with their target RNAs. Two classes of guide snoRNAs are known: (1) box C/D RNAs that guide 2′-O-methylation, and (2) box H/ACA RNAs that guide pseudouridylation. Eukaryal box C/D RNAs are characterized by two conserved motifs, box C- and box D-containing consensus sequences RUGAUGA and CUGA, respectively, which are present near the two termini of the RNA. Imperfect copies of these motifs (box C′ and D′) are frequently present internally. Eukaryal box C′ and D′ sequences are often degenerate and difficult to identify.11 A stretch of 10–21 nucleotides 5′ of box D and sometimes box D′ pair with their target RNA. The target residue is chosen by a strict base-pairing rule: the ribose of the target nucleotide that base pairs with the fifth nucleotide 5′ of the box D or D′ in the guide, is specifically methylated. In addition to the guide RNA, the box C/D snoRNP is composed of four core proteins—Nop56p, Nop58p (Nop5p), 15.5-kDa (Snu13p) and fibrillarin (Nop1p), the last one functioning as a methyltransferase.4–6,8

Archaea also contain both box C/D and box H/ACA snoRNA-like RNAs (sRNAs).7–10,12–17 In addition to rRNAs, archaeal sRNAs can also target tRNAs for modification. The overall structures of archaeal and eukaryal box C/D RNAs are similar. However, unlike in Eukarya, box C′ and D′ in Archaea are well conserved.8,18,19 Archaeal proteins that form the box C/D RNP complex are aNop5p (a single homolog to eukaryal Nop56/58p), L7Ae (homolog of 15.5-kDa) and the methyltransferase afibrillarin (aFib).4,8,20–25 L7Ae binds to K-turn and K-loop, the folded RNA structures found in both box C/D and H/ACA RNAs.10,26–29 The canonical K-turn consists of a 3-base bulge flanked on its 5′ side by a normal base-paired canonical stem (C stem) and on its 3′ side by a non-canonical stem (NC stem) containing tandem, sheared G•A base pairs.27,30 All known modified versions of K-turns contain an asymmetric bulge between these two stems (www.dundee.ac.uk/biocentre/nasg/kturn/index.php). The archaeal C′/D′ motif frequently forms a K-loop where the C stem is replaced by a loop.28 L7Ae initiates a stepwise assembly of box C/D sRNP by binding to the K-turn/K-loop, which is followed by aNop5p and aFib.22,25 Several methylationcompetent in vitro archaeal systems using box C/D sRNAs and recombinant core proteins have been established.22,25,31–33

Many computational methods have been developed to predict the presence of box C/D and box H/ACA sRNAs in the genomes of different organisms.34–36 These predictions are based mainly on the characteristic structural features of the guide sRNAs. The presence of some of these sRNAs has been experimentally verified by Northern analyses or primer extension reactions. Experimental approaches have also been used to identify small non-coding RNAs.37,38 Various small non-coding RNAs have been identified in several Archaea using these computational and experimental approaches.13,14,16,39–45 Interestingly, the intron of pretRNATrp of the haloarchaeon Haloferax volcanii and certain other Euryarchaeota has been shown to act as a guide RNA.12,32,33,46 The intron bears all the characteristics of a box C/D RNA. It guides in trans, 2′-O-methylations of two residues in intron-containing pre-tRNATrp that ultimately become C34 (wobble position) and U39 residues of the spliced tRNA.33,47 This archaeal pre-tRNATrp is a unique case where both the target and guide RNAs are present in the same transcript. The 2′-O-methylations of C34 of intron-containing precursors of the elongator tRNAMet in Methanocaldococcus jannaschii and different Pyrococcus species have also been predicted to be guided by box C/D RNAs, sR8 and sR49, respectively.39,46 However, these guide RNAs are not part of the tRNA intron. Homologs of Pyrococcus sR49 have also been predicted in Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum, but such homologs were unknown in H. volcanii.46 None of these guide RNAs have been experimentally shown to guide the modification in full-size pre-tRNAMet.

In this study, we show that 2′-O-methylation of the C34 residue in H. volcanii elongator tRNAMet is guided by a unique box C/D sRNA that differs both in size and structure from its homologs. Strikingly, the box C′/D′ motif of this H. volcanii guide RNA does not form either the classical K-turn or K-loop in spite of having two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in its non-canonical stem. However, this atypical C′/D′ motif remains capable of forming a stable complex with L7Ae protein. This guide sRNA, named sR-tMet (sRNA for tRNAMet) here, was predicted from sequence analyses to be a 73-nucleotide long RNA. However, the actual size of H. volcanii sR-tMet inside the cell was experimentally found to be 124 nucleotides, whereas it is 73–75 nucleotides in other haloarchaea. The 51-base extension in H. volcanii is determined to be present on the 3′-end of the RNA and it does not interfere with sR-tMet activity in vitro. This extension is present in the precursor but not in mature sRtMet of Halobacterium sp, suggesting a different 3′-end processing of sR-tMet in these two closely related organisms. This report of an sRNA differing in size from its prediction, raises questions about the actual sizes of many other sRNAs predicted from sequence analyses, but not experimentally verified in vivo.

Results

Prediction of a putative box C/D guide RNA for ribose-methylation of wobble residue C34 in intron-containing precursor of H. volcanii elongator tRNAMet.

Wobble residue C34 in the anticodon of H. volcanii elongator tRNAMet is 2′-O-methylated. However, neither a specific methyltransferase nor a guide box C/D RNA for this modification has yet been identified. We reasoned that this 2′-O-methylation might be guided by a box C/D RNA and the modification occurs in intron-containing precursor of this tRNA, as is the case for tRNATrp. The rationale for such a conclusion is four-fold: (1) C34 of only elongator tRNAMet and tRNATrp of H. volcanii are 2′-O-methylated, whereas C34 of other tRNAs are either unmodified or contain N4-acetyl C;48,49 (2) the genes for these two tRNAs contain introns, whereas genes encoding other C34 containing tRNAs do not have introns;50–52 (3) the introns in the two tRNA genes are present at the same positions, i.e., between residue 37 and 38 of the mature tRNA; and (4) box C/D RNAs have been predicted to guide 2′-O-methylation of C34 of intron-containing precursors of elongator tRNAMet in some Euryarchaeota but not in haloarchaea. Indeed a search by Pyrococcus sR49 or by M. jannaschii sR8 sequence does not reveal a homolog in H. volcanii or in other haloarchaea. Although the intron of H. volcanii pre-tRNAMet is 75 bases long,51 much larger than the corresponding introns in other non-halophilic Archaea, it does not have box C/D RNA like features. Computational approaches led us to identify a 73-base sequence in the H. volcanii genome (position 2163553–2163625), named sR-tMet here, that has all the features of a potential box C/D guide RNA for 2′-O-methylation of C34 of the intron-containing elongator pre-tRNAMet. The 15 bases preceding its box D can pair with properly positioned 15 bases in the target region for C34 modification in the pre-tRNAMet. sR-tMet homologs are also found in other haloarchaea (Fig. 1A). Sequences of several regions within sR-tMet of the four haloarchaeal homologs shown in the figure are identical, specifically, a 22-base region at the 3′ end and a 12-base region at the 5′ end. However, the genomic context of sR-tMet in different haloarchaea does not show any conservation (data not shown). The region between box C′ and D′ of H. volcanii sR-tMet is significantly larger than the comparable regions in M. jannaschii sR8 and Pyrococcus sR49 (Fig. 1B). The box C′/D′ motif of both M. jannaschii sR8 and Pyrococcus sR49 can form a K-loop. However, box C′ and D′ and the sequence between them in H. volcanii sR-tMet does not fold into a typical K-turn or a K-loop, in spite of having two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in the noncanonical stem. Thus the box C′/D′ motif, probably folds into an alternative K-turn in vivo. Furthermore, the D-guide/spacer (between C′ box and D box) of H. volcanii sR-tMet is comprised of 15 residues, whereas the same region of M. jannaschii sR8 and Pyrococcus sR49 contains 12 residues each (Fig. 1B).

Figure 1.

Figure 1

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H. volcanii sR-tMet and its homologs. (A) Multiple sequence alignment of sR-tMet gene and its flanking regions in H. volcanii (Hv), Halobacterium sp NRC-1 (HN), Natronomonas pharaonis (Np), and Haloarcula marismortui (Hm). Position 1 denotes the 5′ end of the RNA and negative numbers indicate the positions 5′ to this end. Schematic representation of the box sequences and regions between the boxes are shown above the alignment. (B) Sequences and predicted secondary structures of H. volcanii sR-tMet, P. abyssi sR49 and M. jannaschii sR8 RNAs. Different boxes are designated. Pairings of D-guide/spacer regions with complementary target regions of pre-tRNAMet are shown, and target nucleotides are circled. Arrows indicate the position of the 5′ exon-intron junction in the target pre-tRNA.

sR-tMet RNA is present in haloarchaea, but it is substantially larger in H. volcanii than its other haloarchaeal homologs.

Total RNA from H. volcanii, Halobacterium sp NRC-1, Natronomonas pharaonis, Haloarcula marismortui, and Haloarcula quadrata was hybridized to a primer that is complementary to the 22 base identical sequence region at the 3′ end of sR-tMet of haloarchaea (Fig. 2). The results suggest that sR-tMet RNA is present in these different halophiles in sufficient quantities to be visualized by RNA gel blot analysis. Interestingly, while all other halophilic sR-tMet RNAs are of the predicted size of about 73–75 bases, that of H. volcanii is substantially larger. It migrated to the position of 5S rRNA (about 125 bases) while that of the other species migrated to the position of class I tRNAs (about 75–80 bases); this suggests that sR-tMet of H. volcanii is larger than the size predicted from sequence analyses. Therefore, based on the genomic sequence of H. volcanii (Fig. 1A), sR-tMet is expected to have an extension either at one or both ends.

Figure 2.

Figure 2

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sR-tMet RNA is present in several haloarchaea. RNA gel blot of total RNA separated by 6% denaturing PAGE is hybridized to 5′ 32P-labeled oligonucleotide complementary to the 22-base sequence common to 3′ region of sR-tMet RNAs of haloarchaea (position 54–75 in Fig. 1A). Lanes 1–5 represent RNA of H. volcanii, Halobacterium sp NRC-1, Natronomonas pharaonis, Haloarcula marismortui, and Haloarcula quadrata, respectively. The asterisk marks the position of class I tRNAs (size about 75 bases).

H. volcanii sR-tMet RNA has a 3′ extension beyond the bioinformatic prediction.

Primer extensions were done to map the 5′ ends of haloarchaeal sR-tMet RNAs (Fig. 3B) using the same common primer and total RNA from the same 5 haloarchaea used for Northern analysis (Fig. 2). These results determined the 5′ ends of the sR-tMet RNAs to be at the predicted G shown at position 1 in Figure 1A. As expected from the sequence alignment, the extension product of H. marismortui is two bases larger than the products of H. volcanii, Halobacterium sp and N. pharaonis. The sequence of H. quadrata sR-tMet is not available. However, based on the data of Figures 2 and 3B, its size should be the same as that of H. marismortui sR-tMet and it also contains sufficiently similar sequence at its 3′ end for it to bind to the common primer.

Figure 3.

Figure 3

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The 5′ ends of haloarchaeal sR-tMet RNAs agree with predicted sequences. (A) A linear representation of H. volcanii sR-tMet and positions of primers R1–R4 used for extension studies. Solid line indicates predicted 73-base RNA and dashed line indicates its 3′ extension. Numbers next to arrows indicate the position in the RNA up to which the primers hybridize. Numbers in parentheses indicate the equivalent positions in Figure 1A. R1 is common to different haloarchaea and was also used for Northern analysis (Fig. 2). (B) sR-tMet RNAs of haloarchaea have similar 5′ ends. 5′ 32P-labeled R1 primer was used in reverse transcriptase extension reactions with total RNA from H. volcanii (HV), Halobacterium sp NRC-1 (NRC1), N. pharaonis (NP), H. marismortui (HM) and H. quadrata (HQ). The products were separated by 12% denaturing PAGE. Sequencing reactions of an unrelated DNA are used as size markers. As predicted (see Fig. 1A), products of 73 bases are observed for the first 3 organisms and of 75 bases for the last 2 organisms. (C) H. volcanii sR-tMet has an extension on the 3′ end beyond its predicted length. the primer extension study was done as in B using R2, R3 and R4 primers and H. volcanii total RNA. As predicted (see Fig. 1A), products of 73, 79 and 93 bases were observed with R2, R3 and R4, respectively. Sequencing reactions of an unrelated DNA are used as size markers.

The 5′ end mapping results shown in Figure 3B suggested that mature H. volcanii sR-tMet would have a 3′ extension beyond its predicted size of 73 bases. To confirm this, primer extensions were done using H. volcanii total RNA and three different primers (Fig. 3C). The binding sites for these primers extend up to the predicted 3′ end (position 73 in Fig. 3A) or to regions beyond this (positions 79 or 93 in Fig. 3A). The extension product from the primer extending up to position 93 would be obtained only if there was an extension beyond the predicted 3′ end. As expected, cDNA products were observed in all cases, reinforcing the fact that H. volcanii sR-tMet bears a 3′ extension.

The 3′ end of H. volcanii sR-tMet was mapped by an RNase protection assay using a 3′ end-labeled 155 base RNA probe that could hybridize to 37 bases at the 3′ end of the predicted sequence of H. volcanii sR-tMet (Fig. 4A). An 88-base RNase-protected fragment was observed in this assay. This suggested that the exact 3′ end of H. volcanii sR-tMet extends 51 bases beyond its predicted 3′ end, i.e., up to position C127 in Figure 1A. Therefore, the size of mature H. volcanii sR-tMet is 124 bases, which includes a 51-base 3′ extension beyond the sR-tMet RNAs of other haloarchaea. An RNase protection assay was also done to map the 3′ end of Halobacterium sp NRC-1 sR-tMet (Fig. 4B), and the major species was found to be 74 bases, terminating at T76 of Figure 1A.

Figure 4.

Figure 4

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A 3′ extension is present in sR-tMet RNA of H. volcanii but not in that of Halobacterium. (A) A 3′ end-labeled 155-base RNA probe was used in an RNase protection assay to map the 3′ end of H. volcanii sR-tMet. The probe can hybridize to sR-tMet starting at position G39 in Figure 1A. Separation was done on a 12% denaturing gel. Undigested and partial alkaline hydrolyzed probes were run in lanes 1 and 3, respectively. RNase-digest of probe hybridized to total RNA of H. volcanii is shown in lane 2. Lane 4 contains size markers prepared by I2/EtOH cleavage of an unrelated 5′ end-labeled RNA prepared with ATPαS. The 88-base RNase protected fragment determined 3′ end of the RNA to be at C127 of Figure 1A. (B) Similarly, a 3′ end labeled 58-base RNA was used to determine the 3′ end of Halobacterium sp NRC-1 sR-tMet RNA. The probe can hybridize to this RNA starting at position G54 in Figure 1A. Undigested and partial alkaline hydrolyzed probes were run in lanes 1 and 4, respectively. An RNase-digest of the probe in the presence and absence of total Halobacterium RNA are shown in lanes 3 and 2, respectively. As a size marker, a 5′ end-labeled 19-mer oligonucleotide is shown in lane 5. The 23-base RNase-protected fragment determined the 3′ end of the RNA to be at T76 of Figure 1A.

Large transcripts are processed to produce mature termini of haloarchaeal sR-tMet.

An examination of haloarchaeal sRtMet sequences just before the +1 position and just after 3′ termini of these RNAs (Fig. 1A) failed to show significant sequence matches with predicted haloarchaeal promoters and terminators.53 Therefore, it is expected that these RNAs are processed from the precursors containing extensions on both 5′ and 3′ ends. To confirm this, reverse transcriptase reactions followed by PCR were done using total RNA from cells of Halobacterium sp NRC-1 as template (Fig. 5). The results suggest that the precursor of the 73–74-base sR-tMet has at least an 80 base extension on its 5′-side and a 50–51 base extension on its 3′ side. This 3′ base extension is observed in the precursor but not in the mature sR-tMet RNA of Halobacterium sp NRC-1. However, this extension is retained in the mature sR-tMet in H. volcanii (see Fig. 4). A sequence of 32 bases (positions 54–86, Fig. 1A) is identical for sR-tMet RNAs of these two species. However, mature Halobacterium sp NRC-1 sR-tMet is formed by a cleavage (endo- or exonucleolytic) within this sequence (between position 75 and 76, Fig. 1A), whereas the site of cleavage in H. volcanii is downstream of this position.

Figure 5.

Figure 5

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Precursor of Halobacterium sR-tMet contains extensions on both 5′ and 3′ ends. Primer A was used in reverse transcriptase reactions using total RNA from Halobacterium sp NRC-1. A cDNA product was used as template for PC R and the products were separated by 6% native PAGE. The schematic on the left represents sR-tMet (solid line) and extensions (dashed lines) on its two sides. Arrows mark the primers used in the reactions. Starting positions of the primers are indicated. M, size markers. Primer pairs for PC R are indicated above each lane, and the expected size of the PC R product is indicated below each band. Negative controls using total RNA in PC R did not yield any product (not shown).

H. volcanii sR-tMet with or without its 51-base extension, can guide ribose methylation of C34 of pre-tRNAMet in vitro.

The predicted structure of the 124-base H. volcanii sR-tMet box C/D RNA is shown in Figure 6A. To determine if H. volcanii sR-tMet can guide the modification of C34 of pre-tRNAMet and whether its 3′ extension plays any role in the reaction, a series of in vitro modification assays were done using sR-tMet RNAs of 73, 105, and 124 nucleotides (Fig. 6C). The 73-base sequence is the size of the predicted RNA with no 3′ extension. The 124-base transcript has full-size 3′ extensions, as is present in vivo. The intermediate sized 105-base transcript was used to determine whether a shorter 3′ extension can also function in in vitro assays. This 105-base RNA can form a short (16-base) stem-loop structure instead of the large stem-loop formed in full-sized RNA. These three guide RNAs were independently used with radiolabeled pre-tRNAMet (Fig. 6B) in modification reactions with recombinant M. jannaschii core proteins (L7Ae, aNop5p and aFib) and AdoMet. Following TLC separation of the RNase T2 digested samples, a CmAp dinucleotide was observed in all cases (Fig. 6C), and the presence or absence of a 3′ extension in sRtMet RNA did not affect the amount of modification. This is consistent with the observation that the 3′ extension is not part of the basic structure of box C/D RNA and the extension is not present in sR-tMet of other haloarchaea. Radiolabeled CmAp dinucleotide was also observed when an H. volcanii cell extract was used as the source of sRNPs in reactions (Fig. 6D). This methylation activity in cell extracts is consistent with methylation by in vitro assembled sRNPs.

Figure 6.

Figure 6

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In vitro transcribed guide sR-tMet and target pre-tRNAMet of H. volcanii can function in in vitro 2′-O-methylation reactions to specifically modify C34. (A) Sequence and predicted secondary structure of 124-base sR-tMet transcript. It contains a 51-base 3′ extension beyond the standard box C/D RNA structure. The 3′ ends of the 105-base and 73-base transcripts are marked by arrows. The 105-base transcript can have a shorter stem-loop structure covering position 83–98. The C, D, C′ and D′ boxes are enclosed and labeled. Guide sequence and guide residue are indicated by a line and an asterisk, respectively. (B) Sequence and predicted secondary structure of pre-tRNAMet. Anticodon nucleotides (CAU) are circled and the target sequence is indicated by a line. Target residue C, which is marked with an asterisk, is considered to be at position 34 in the standard numbering system for mature tRNAs. Exon-intron junctions, indicated by arrows, are located within the bulge-helix-bulge structure required for archaeal pre-tRNA splicing. (C) [α-32P]ATP-labeled pre-tRNA was either incubated alone (control) or with different sized unlabeled sR-tMet in 2′-O-methylation reactions followed by RNase T2 digestion and TLC analysis; mononucleotide and dinucleotide products are indicated. The appearance of radiolabeled CmAp indicates the 2′-O-methylation of target C in the pre-tRNA. (D) TLC analyses as in C, except using H. volcanii cell-extracts instead of guide RNA and recombinant proteins in the reaction. (E) TLC analyses as in C after methylation reaction of [α-32P]UTP-labeled 5′ half of pre-tRNAMet (positions 1–74 in B) and its U23C mutant with 73-base unlabeled sR-tMet.

The specificity of sR-tMet-guided modification of C34 (at the wobble position of anticodon) was determined by using the 5′ half of pre-tRNAMet as the target RNA. This truncated sequence has a 5′-CAU-3′ at two positions (21–23 and 35–37 in Fig. 6B). We changed U23 to C in this sequence and used the resulting mutant as well as the non-mutated [α-32P]-UTP labeled RNAs as substrates in our sR-tMet guided modification reactions (Fig. 6E); a CmAp dinucleotide was observed in both cases. The amount of modification was also the same in both cases, and these data prove that the modification occurs only at position 34.

The D′-guide/spacer (between C box and D′ box) is probably not functional, as this region varies among different haloarchaea (Fig. 1A). Furthermore, it is ten nucleotides long, which is less than the 12 base optimum size for an archaeal guide/spacer sequence.54 Nevertheless, we still checked whether this region can function as a guide in vitro. To do this we used two different anti-sense RNAs in our in vitro methylation reactions. These target RNAs were a 20-mer (5′-GGA CAA GCG GUG CCG GAA GG-3′) and a 23-mer (5′-GGC AUU CGG AGC GGU GCC GGC UG-3′) and contained sequences (underlined) complementary to positions 13–22 and 13–27, respectively, of sR-tMet, as shown in Figure 6A. No modification was observed in either case (data not shown). The reason for testing two different anti-sense RNAs is that there are 2 CCGA in H. volcanii sRtMet (positions 23–26 and 28–31, Fig. 5A) that potentially can function as a D′ box. However, we predict that the first CCGA (23–26) is the actual D′ box of the RNA because of its conservation in other haloarchaea (see Fig. 1A).

The haloarchaeal sR-tMet box C′/D′ motif apparently does not form a typical K-turn or K-loop.

The C′/D′ motif of H. volcanii sR-tMet does not fold into a typical K-turn or a K-loop (Fig. 1B and 6A). Although it contains two tandem, sheared G•A base pairs and a pyrimidine-pyrimidine pair in its non-canonical stem, it lacks an internal asymmetric loop typical of a K-turn. To test whether this governs the inability of the D′-guide/spacer to function, two modified versions of sR-tMet were created. One had an insertion of three bases to create a typical K-turn in the box C′/D′ motif, while the other had a deletion of 14 bases to create a typical K-loop in this motif. Both of these modified versions were used as guide RNAs with the above-mentioned 20-mer and 23-mer target RNAs in in vitro modification reactions. Neither of these target RNAs showed modification (data not shown). This suggests that the absence of a K-turn or K-loop in the C′/D′ motif is not the only reason that the D′ guide region is unable to participate in the modification reaction.

Box C/D RNAs containing both box C/D and C′/D′ motifs and either a K-turn or K-loop in the box C′/D′ motif have been shown to form 2 sRNP complexes with increasing concentrations of L7Ae.25,28,29,31,47 Therefore, sR-tMet RNA was mixed with increasing concentrations of the L7Ae protein to determine if the box C′/D′ motif lacking a K-turn or K-loop can also bind the protein. Gel-shift analyses of assembled RNPs suggest that at lower concentrations of protein, only one ribonucleoprotein (RNP) is formed (RNP1 in Fig. 7). With increasing concentrations of protein, a new RNP of slower mobility (RNP2) appears, which increases with a concomitant decrease of RNP1. These results suggest that H. volcanii sR-tMet can bind 2 L7Ae proteins, most likely one each to the box C/D and C′/D′ motifs. Perhaps the box C′/D′ motif in this case forms an alternative K-turn that can bind L7Ae. The significance of this lies in the fact that to our knowledge, this is the first report of L7Ae binding to an RNA motif that lacks a typical K-turn or K-loop structure.

Figure 7.

Figure 7

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L7Ae protein can form two RNP complexes with H. volcanii sRtMet. Radiolabeled H. volcanii sR-tMet (0.56 pmol) was incubated with increasing concentration of M. jannaschii L7Ae protein and resolved by 4% native PAGE. The two complexes obtained are labeled as RNP1 and RNP2.

Discussion

Ribose-methylation of wobble residue (C34) of haloarchaeal elongator pre-tRNAMet is guided by a unique box C/D sRNA.

There is paucity of guide sRNAs in haloarchaea, although not in other Archaea. Previously only one box C/D guide sRNA, the intron of pre-tRNATrp, has been identified in haloarchaea.12,32,33,46 This is an atypical guide sRNA present in haloarchaea and some other Euryarchaeota. Unlike all other box C/D guide RNAs, this is the only known case where both guide and target RNA are present in the same transcript. Here we present the second box C/D guide RNA in haloarchaea. This newly identified box C/D guide RNA (named sR-tMet) is distinct from its target RNA, the intron-containing elongator pre-tRNAMet, as is the case for all other known box C/D guide RNAs. sR-tMet was shown to be present in all five of the haloarchaeal species that we tested. It can participate in our previously described in vitro system to produce 2′-O-methylation of a target residue using in vitro transcribed H. volcanii guide and target RNAs and M. jannaschii core proteins.33,47 Most of the other established in vitro 2′-O-methylation systems have used small oligonucleotides as target RNAs to assess nucleotide modification. The system described herein is the only functional system known that uses separate natural-sized RNA components: sR-tMet as guide and pre-tRNAMet as target.

The 3′ extension of sR-tMet is present in H. volcanii but not in other haloarchaea.

H. volcanii sR-tMet has a 51-base extension on the 3′ end, which is absent in the other mature haloarchaeal sR-tMet RNAs. In vitro 2′-O-methylation reactions using guide RNAs with or without this extension do not show any difference (Fig. 6C). These results along with the absence of this extension from other haloarchaea, suggest that the 51-base extension does not function in 2′-O-methylation. It may play some other role that is specific to H. volcanii but not to other haloarchaea. The extension is present in the precursor of sR-tMet of Halobacterium (Fig. 5). Furthermore, a 32-nucleotide sequence (position 54–86 in Fig. 1A) is identical for sR-tMet of Halobacterium and H. volcanii. However, cleavage occurs within this identical sequence in the former case but outside this sequence in the latter case. This raises a question about differential processing of similar transcripts in closely related organisms. We are working to delete this extension from H. volcanii genome to determine whether it (1) is redundant, (2) affects the processing of sR-tMet RNA, or (3) has some other role.

To our knowledge, no other box C/D guide RNA with a substantial 3′ extension has yet been identified. Two termini of box C/D RNAs mostly pair to form the C stem of the K-turn of the C/D motif. Therefore, 2 ends of the predicted box C/D guide RNAs are routinely indicated as base-paired regions. H. volcanii sR-tMet RNA is an example where the size of a predicted guide RNA differs significantly from its experimentally determined counterpart. Therefore, absence of an RNA in the predicted size range does not necessarily mean that the RNA is absent from the cell.

Structure of haloarchaeal sR-tMet differs from other euryarchaeal homologs.

M. jannaschii sR8 and Pyrococcus sR49 have been predicted to guide the 2′-O-methylation of C34 of elongator pre-tRNAMet,39,46 the same modification guided by haloarchaeal sR-tMet. Potential homologs of Pyrococcus sR49 were identified in certain Euryarchaeota but not in H. volcanii.46 We also could not identify haloarchaeal homologs by searches using sequences of M. jannaschii sR8 and Pyrococcus sR49. Therefore, we searched the H. volcanii genome using a 15-mer sequence that could have a box D preceded by a potential guide sequence for modification of residue C34 in intron-containing pre-tRNAMet. The search resulted in haloarchaeal sR-tMet, which significantly differs from its other euryarchaeal homologs, both in sequence and secondary structure (see Fig. 1). During the course of our study Grosjean et al.55 have independently predicted the sequence of this guide RNA in the H. volcanii genome.

H. volcanii sR-tMet, M. jannaschii sR8 and Pyrococcus sR49, as well as their other homologs, use their D-guide/spacer region for modification of C34 of elongator pre-tRNAMet. Thus far, we have not been able to find an rRNA or tRNA target sequence for the D′-guide/spacer region of H. volcanii sR-tMet. Sequence heterogeneity of this region among different haloarchaea (see Fig. 1A) suggests that this region may not have a functional target. Our inability to modify appropriate oligonucleotides in vitro is consistent with a D′-guide/spacer region being non-functional. Sequence differences in this D′-guide/spacer region of Pyrococcus sR49 also suggested absence of a target for this region in sR49.46 However, M. jannaschii sR8 is proposed to be a dual guide RNA, D-guide/spacer region for C34 of elongator pre-tRNAMet and D′-guide/spacer region for G900 of 23S rRNA (http://lowelab.ucsc.edu/snoRNAdb/Archaea/Mja-annote.html).39

The internal box C′/D′ motif of H. volcanii sR-tMet contains two tandem, sheared G•A base-pairs and a pyrimidine-pyrimidine pair in its non-canonical stem, but does not show typical features of either a K-turn or a K-loop (Fig. 1B). This differs from the box C′/D′ motifs of M. jannaschii sR8 and Pyrococcus sR49, both of which contain K-loops (Fig. 1B). Archaeal L7Ae protein can bind to both a K-turn and a K-loop in box C/D and box H/ACA RNAs.10,16,23,27–29,56–58 Titration of guide RNAs that contain both box C/D and C′/D′ motifs with K-turn/K-loop, show formation of two distinct RNP complexes with increasing concentrations of L7Ae.25,28,29,31,47 H. volcanii sR-tMet can also form two distinct RNP complexes with increasing concentrations of L7Ae (Fig. 7), in spite of the lack of a typical K-turn/K-loop in its C′/D′ motif. This suggests that L7Ae binds to a modified K-turn in the box C′/D′ motif of haloarchaeal sR-tMet. This unusual C′/D′ motif shows some similarity to SECIS elements in 3′ UTRs of eukaryal mRNAs, which is required for cotranslational insertion of selenocysteine in their proteins.59,60 In vitro, archaeal L7Ae protein can bind SECIS RNA.59,60 However, the unusual C′/D′ motif of H. volcanii sR-tMet cannot have a role in the insertion of selenocysteine in proteins, as Halobacterium and some other Archaea do not encode the selenocysteine insertion machinery.61 Presently we are working to characterize this unusual C′/D′ motif and its binding to L7Ae and other proteins, which may provide new insights into the structure-function relationship of this RNA motif.

Materials and Methods

DNA sequence analysis.

Partial sequence of the H. volcanii genome was available at the University of Scranton site at the time of our original search for sR-tMet, and this sequence was downloaded from there. Microsoft Word was used to search the genome for a 15-mer sequence CCCTATGAGTGCTGA and its complement, allowing at the most two mismatches: one at position eleven and another one within the last four bases. The first 11 bases of the 15-mer are complementary to the potential target sequence for 2′-O-methylation of H. volcanii elongator pretRNAMet C34 and the last 4 bases correspond to a potential D box. Flanking sequences of the matches were manually searched for potential to form a box C/D sRNA. Multiple-sequence alignments of the selected sequence with its haloarchaeal homologs were done with the Clustal W62 program using sequence data from the UCSC Archaeal Genome Browser (archaea.ucsc.edu/).

Organisms and growth.

All halophiles were grown at 42°C. Composition of growth media for H. volcanii, H. quadrata and for all others are according to Gupta,48 Oren et al.63 and Robb et al.,64 respectively.

Cloning and production of recombinant proteins.

Initially genes of H. volcanii L7Ae, aNop5p and aFib proteins were cloned to produce recombinant proteins by overexpression in E. coli. However, these proteins did not show any activity. This may be due their inactivation in low salt concentrations. Most halophilic enzymes are inactivated when Na+ or K+ concentrations in the solution decreases to less than 2M.65 Therefore, we prepared and used recombinant M. jannaschii proteins as described before.25,33

RNase protection assay.

To determine the 3′ ends of the RNAs, complementary RNA riboprobes were prepared by in vitro transcription of a T7 RNA polymerase promoter containing PCR products using primers based on the genomic sequences. The riboprobes were 3′ end-labeled for use in RNase protection assays following standard procedures.66 Briefly, labeled riboprobe was combined and precipitated with DNase-treated total RNA. The precipitate was dissolved in 30 µl of hybridization buffer (40 mM PIPES, pH 6.8, 1 mM EDTA, 0.4 M NaCl, 80% deionized formamide), heated at 85°C and further incubated for 12 h at 45°C–50°C. Then 300 µl of RNase digestion buffer (300 mM NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA) and 1 µl of appropriate dilution of an RNase mixture were added to the sample and incubated for 60 min at 30°C. The RNase mixture was prepared by mixing equal volumes of RNase A (100 µg/ml) and RNase T1/T2 prepared according to a published procedure.67 Appropriate dilution of the RNase mixture was determined by titrating various dilution. After digestion, 20 µl of 10% SDS and 10 µl of Proteinase K (10 mg/ml) was added and incubated at 37°C for 30 min. The RNA was then extracted with phenolchloroform, precipitated and separated on a 12% denaturing polyacrylamide gel.

Other procedures.

Total RNA was isolated from different halophiles using Tri reagent (Molecular Research Center) followed by DNase treatment. MMLV reverse transcriptase (Promega) was used according to the manufacturer's protocol for primer extension studies and the preparation of cDNA for PCR. General procedures for DNA template construction and in vitro transcription have been described previously.33,68 Electrophoretic mobility shift assays followed established procedures.33 In vitro 2′-O-methylation reactions using recombinant M. jannaschii proteins and H. volcanii cell extracts, and detection of modified nucleotides by thin layer chromatography, were done following our previous methods.33,47 Partial alkaline hydrolysis of RNA was also done as described earlier,48 and preparation of RNA with ATPαS and its I2/EtOH cleavage was according to a standard protocol.69 Standard procedures66 were used for other molecular biological work. Sequences of the oligonucleotides used in this study are available upon request.

Acknowledgments

We thank Shiladitya DasSarma, Mike Dyall-Smith, Thomas Alton and Richard Shand (deceased) for providing the strains of Halobacterium sp NRC-1, Haloarcula marismortui, Haloarcula quadrata and Natronomonas pharaonis, respectively, Stu Maxwell for various suggestions and review, Mike Madigan for critical reading of the manuscript, and Julie Maupin-Furlow, Sanjay Singh and Priyatansh Gurha for helpful discussions. This work was supported by National Institutes of Health Grant GM55045 to R.G.

Abbreviations

snoRNA

small nucleolar RNA

snoRNP

small nucleolar ribonucleoprotein

sRNA

snoRNA-like RNA

aFib

afibrillarin

C stem

canonical stem

NC stem

non-canonical stem

RNP

ribonucleoprotein

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