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Journal of Bacteriology, November 2008, p. 7308-7313, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00820-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Box C/D RNA-Guided 2'-O Methylations and the Intron of tRNA^Trp Are Not Essential for the Viability of Haloferax volcanii

Archi Joardar, Priyatansh Gurha, Geena Skariah, and Ramesh Gupta^*

Department of Biochemistry and Molecular Biology, Southern Illinois University, Carbondale, Illinois 62901-4413

Received 10 June 2008/ Accepted 23 August 2008

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Deleting the box C/D RNA-containing intron in the Haloferax volcanii tRNA^Trp gene abolishes RNA-guided 2'-O methylations of C34 and U39 residues of tRNA^Trp. However, this deletion does not affect growth under standard conditions.

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Ribose methylation (2'-O methylation) and pseudouridylation (isomerization of uridine to pseudouridine) of stable RNAs, e.g., rRNAs, tRNAs, and small or sno-like RNAs (snRNAs), can occur by the activity of stand-alone proteins in all organisms and also by the activity of ribonucleoproteins (RNPs) in the Eukarya and Archaea (7-10, 14, 16, 19, 22, 26, 31). The RNPs contain guide RNAs called small nucleolar RNAs (snoRNAs) in the Eukarya and sRNAs in the Archaea. Box H/ACA and box C/D guide RNAs, named after specific conserved sequences, direct the pseudouridylations and 2'-O methylations, respectively, of the specific residues in the target RNAs. Box C/D RNAs contain a conserved box C/D motif near their termini and a less-conserved box C'/D' internally. Guide sequences, generally 10 to 21 nucleotides in length, located 5' to the D and D' boxes can pair with corresponding sequences in the target RNA. The residue in the target RNA that pairs with the fifth base on the 5' side of the D or D' box gets 2'-O methylated. Eukaryal guide RNAs associate with fibrillarin (a methyltransferase), Nop56p, Nop58p, and 15.5K (Snu13p) proteins to form core snoRNP complexes. The corresponding archaeal proteins are fibrillarin, aNop5p (homologous to both Nop56p and Nop58p), and L7Ae (15.5K homolog).

The intron-containing pre-tRNA^Trp of Haloferax volcanii (Fig. 1A) and certain other members of the Euryarchaeota is unique in having both guide and target sequences in the same RNA molecule (6, 20, 25). The intron can function as box C/D sRNA in trans while still part of the pre-tRNA^Trp or in free excised form (in both linear and circular versions) (23, 25). The D and D' guide sequences of the intron can sequentially guide 2'-O methylations of target residues C34 and U39 (Cm34 and Um39 in Fig. 1B), respectively (24, 25). These residues are located in the two exon regions of the pre-tRNA^Trp (Fig. 1A). The target sequences around C34 and U39 that pair with the corresponding guide sequences overlap the 5' and 3' exon-intron junctions, respectively (25). This necessitates that the methylation reactions occur in the intron-containing pre-tRNA and precede splicing of the intron, since the regions near the two termini of the intron form part of the target sequences. Several intron-dependent modifications of nucleosides have been identified in eukaryal tRNAs (2, 5, 11). However, unlike the intron-dependent modifications in the tRNA^Trp of H. volcanii, which are RNA guided, these are produced by protein-only enzymes. The aim of this work is to determine the viability of H. volcanii cells and the status of the C34 and U39 modifications when the intron of the tRNA^Trp gene has been deleted. These studies should yield insight into the functional significance of these two modifications.

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FIG. 1. Primary sequence and predicted secondary structure of the H. volcanii pre-tRNATrp and tRNATrp. (A) The 103-base intron containing pre-tRNA. The anticodon bases (CCA) are in large letters. The exon-intron junctions are indicated by arrows. Boxes C, D, C', and D' are enclosed and designated. Complementary guide and target sequences are designated by the thick (box C/D) and thin (C'/D') lines. Target C and U nucleotides are numbered 34 and 39, respectively. Complementary guide (lowercase) and target (uppercase) nucleotide pairs (g117:C34 and a70:U39) are shown in black squares (C/D motif) and black circles (C'/D' motif), respectively. The structure is reprinted from reference 25. (B) Mature tRNATrp (12). Numbers are employed according to the standard tRNA numbering system. Standard abbreviations (listed at http://library.med.utah.edu/RNAmods/) for the modified nucleosides are used (10, 17). An arrow indicates the splice junction.

Construction of an H. volcanii strain containing an intronless tRNA^Trp gene. A genomic deletion was created in H. volcanii strain H26 (a pyrE2 strain) by published methods (1). In brief, the HindIII-XbaI fragment of pVT9P11i containing an intronless tRNA^Trp gene (13) was cloned into pTA131 (1). H. volcanii strain H26 transformed with the resulting plasmid was plated on Hv-Ca plates (1). Selected transformants were further grown in Hv-Ca medium and plated on Hv-YPC plates (1) in the presence of 5-fluoroorotic acid. A complete deletion of the 103-base intron in the selected strain (named H26Wi) was determined by PCR, sequencing of the PCR product, and Southern analyses (Fig. 2 and 3). Genomic DNA of strains H26 and H26Wi was used for the PCR. Exon-specific primers generated intron-containing and intronless products with the DNA of strains H26 and H26Wi, respectively (Fig. 2A, lanes 4 and 5). Sequencing of H26Wi product indicated precise deletion of the intron (Fig. 2B). As expected, intron-specific primers showed a product with H26 DNA only (Fig. 2A, lanes 2 and 3). SalI restriction fragments of the DNA of strains H26 and H26Wi, which hybridized with the exon-specific probe, were approximately 1.3 and 1.2 kb, respectively (Fig. 3, lanes 1 and 2). The observed difference between the two strains was due to the deletion of the 103-base intron in strain H26Wi. Hybridization using an intron-specific probe showed a 1.3-kb fragment (intron containing) in strain H26 but none in H26Wi (Fig. 3, lanes 3 and 4), thus reconfirming the absence of intron in strain H26Wi.

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FIG. 2. The tRNATrp gene of H. volcanii strain H26Wi does not contain the intron. (A) PCR products using H26 and H26Wi DNA with either exon-specific or intron-specific primers were resolved by electrophoresis on native 6% polyacrylamide gels. The expected sizes of the products obtained using exon-specific primers 5'-GGGGCTGTGGCCAAGC-3' and 5'-TGGGGCCGGAGGGATTTGAAC-3' with H26 and H26Wi DNA are 177 (containing 103-base intron; lane 4) and 74 (intronless; lane 5) nucleotides, respectively. These primers correspond to the two ends of the pre-tRNA (see Fig. 1A). The expected size of the PCR product of intron-specific primers 5'-TAATACGACTCACTATAGGCTTGGCGCCCGGGA-3' (where the underlined sequence does not hybridize to genomic DNA) and 5'-ATCTCCGGTGGGCACCT-3' with H26 DNA is 119 nucleotides. This product contains 17 extra bases (underlined in the first primer) and 102 bases of intron (since the primer hybridizes to G at position 2, skipping the A at position 1). (B) The PCR product of strain H26Wi with exon-specific primers was sequenced. The arrow denotes the junction between the two exons, indicating precise deletion of the intron (see Fig. 1B).

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FIG. 3. The H. volcanii strain H26Wi genome does not contain intron of tRNATrp gene. The figure shows PhosphorImager analyses of Southern blots of SalI-digested DNA of H. volcanii strains H26 (lanes 1 and 3) and H26Wi (lanes 2 and 4) probed with 32P-labeled (at their 5' ends) primers 5'-TGGGGCCGGAGGGATTTGAAC-3' (3' exon specific; lanes 1 and 2) and 5'-TCAGTATATCAGCTGGAGTGTC-3' (intron specific; lanes 3 and 4). The approximate sizes of the fragments hybridizing to the probes are indicated.

H. volcanii strain H26Wi lacks 2'-O methylations of C34 and U39 in tRNA^Trp. To determine the modification status of tRNA^Trp, 10-ml cell cultures of strains H26 and H26Wi were inoculated with 1.5 mCi of [³²P]phosphoric acid as described before (12). Uniformly labeled total RNA was isolated from the cells by use of TRI reagent (Molecular Research Center) according to the manufacturer's protocol. The RNA was resolved by electrophoresis using a denaturing 6% polyacrylamide gel, and total tRNA was eluted from the gel. Labeled tRNA^Trp was isolated by hybridizing total tRNA to a biotinylated oligonucleotide (biotin-GGGGCCGGAGGGATTTGAACCCCCGATCGACTGAT) bound to streptavidin-containing Dynabeads in the presence of tetraethylammonium chloride (3, 28). Purified tRNA^Trp was separately digested with RNase T2 or nuclease P1, and digests were resolved by two-dimensional thin-layer chromatography on cellulose plates (Cel 300; Macherey-Nagel) by the use of isobutyric acid-0.5 N NH₄OH (5:3 [vol/vol]) for the first dimension and 0.1 M sodium phosphate (pH 6.8)-ammonium sulfate-n-propanol (100:60:2 [vol/wt/vol]) for the second (12). (RNase T2 cuts RNA to produce ribonucleoside 3' monophosphates [Np], except when the nucleotide is 2'-O methylated. Dinucleotide NmNp is produced in such cases. Nuclease P1 produces nucleoside 5' monophosphates [pN].)

RNase T2 and nuclease P1 digests of tRNA^Trp from the intron-containing H26 strain show all modified nucleotides (Fig. 4A), as expected. Identities and thin-layer chromatographic migration of modified nucleotides of tRNA^Trp have been determined previously (12). The presence of CmCp and UmCp (which migrates along with CmUp derived from positions 32 and 33) in RNase T2 digests of H26 RNA (Fig. 4A) reflects the presence of 2'-O-methylated C34 and U39, respectively, in the tRNA^Trp of this strain (see Fig. 1B). Furthermore, the presence of pUm in the nuclease P1 digests also reflects the presence of 2'-O-methylated U39. (The pCm also present in the nuclease P1 digest is not position specific, as it can be derived from 2'-O-methylated C at positions 32, 34, and 56 of the tRNA; see Fig. 1B.) The RNase T2 digest of H26Wi RNA does not contain CmCp (Fig. 4A), indicating the absence of 2'-O methylation of C34 of tRNA^Trp, which in this case is derived from the intronless gene.

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FIG. 4. Intron-dependent sRNA-guided 2'-O-methylated residues are not present in the tRNATrp of H. volcanii strain H26Wi. (A) Thin-layer chromatography of RNase T2 (left panels) and nuclease P1 (right panels) digests of the tRNATrp of strains H26 and H26Wi. Strains are shown in the panels. "p" before or after a nucleoside letter indicates the 5' or 3' phosphate of that nucleoside. "m" indicates 2'-O methylation of the nucleoside preceding it. Arrowheads point to relevant nucleotides or to their former locations when they are absent. See previous work (12) for the identities of other modified nucleotides. (B) Limited alkaline hydrolysis ladders of 3'-end-labeled tRNATrp of strains H26 and H26Wi are shown in lanes 3 and 4, respectively. Numbers to the right indicate the positions of "gaps" in one or both lanes. U-specific reactions of the same tRNATrp samples are represented in lanes 1 and 2. The "bands" corresponding to U29, U33, and U39 are labeled.

We have been able to detect (by quantitation of phosphorimage data) 2'-O methylation levels as low as 1% by the use of thin-layer chromatographic systems (25). The absence of 2'-O-methylated U39 is not very clear in the RNase T2 digests of H26Wi RNA, although the CmUp/UmCp spot in the RNase T2 digests of H26Wi RNA exhibits a level of intensity that is less (i.e., approximately half) than that seen in the H26 RNA (which in this case is therefore labeled CmUp in Fig. 4A). However, the absence of 2'-O methylation of U39 in tRNA^Trp of strain H26Wi was confirmed by the absence of pUm in the nuclease P1 digests of H26Wi RNA (Fig. 4A). These results indicate that the 2'-O methylation of the C34 and U39 in the tRNA^Trp of H. volcanii depends on the presence of the intron in the pre-tRNA. No activity is present in the cells that can lead to 2'-O methylation of these residues in the absence of the intron. H. volcanii contains on average 10 to 18 genome copies/cell during different phases of growth (4). However, in spite of this polyploidy, our data suggest that no functional sRNA or intron-containing copy of the tRNA^Trp gene is present in the cells of strain H26Wi. Furthermore, the presence of all other modified nucleotides (see Fig. 1B) indicates that no other modification of this tRNA requires the presence of the intron.

The absence of 2'-O methylation of the residues at positions 34 and 39 of tRNA^Trp in strain H26Wi was further confirmed by limited alkaline hydrolysis. Unlabeled tRNA^Trp was isolated from H26 and H26Wi cells as described above. The tRNAs were labeled at the 3' end with 5' [³²P]pCp (12). Approximately 100,000 cpm (Cerenkov) of end-labeled tRNA was incubated for 15 min at 90°C in a 6-µl volume containing 20 µg of carrier RNA in 0.05 M sodium bicarbonate buffer (pH 9.0)-1 mM EDTA-7.5 M urea-0.05% each of xylene cyanole and bromophenol blue. Hydrolysis products were separated on 12% sequencing gels, and the gels were subjected to phosphorimaging. U-specific sequencing reactions of the same labeled tRNAs were also done by using hydrazine followed by aniline treatment (12, 21). These U reactions were run in the adjacent lanes and served as markers. The alkaline hydrolysis-generated ladder of the tRNA^Trp of strain H26 shows "gaps" at positions 34 and 39, while "bands" are present at these positions in the tRNA^Trp of intron deletion strain H26Wi (Fig. 4B). This suggests that the residues at these positions are 2'-O methylated in tRNA^Trp derived from the intron-containing gene but are not methylated when the intron is absent. (Alkaline hydrolysis does not occur on the 3' side of 2'-O-methylated residues of RNA.) Both strains contain 2'-O-methylated C32 and hence show "gaps" at position 32 (Fig. 4B, lanes 3 and 4). This methylation does not depend on the presence of the intron in the tRNA.

Both the parent and intron deletion strains showed similar levels of growth in rich media under standard conditions (Fig. 5), and no difference in the colonies on plates was noted (data not shown). No special efforts were made to monitor growth of the two strains under stress conditions or in competition with each other. No apparent difference between the aminoacylation levels of the tRNA^Trp of the two strains was observed in Northern blots under acidic conditions (data not shown) (29). Although the anticodon sequences of tRNA^Trp are part of the identity elements for recognition by tryptophanyl-tRNA synthetases in all three domains of life (15, 27, 30), no effect of any anticodon modification in this recognition has been reported. Our aminoacylation data agree with this.

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FIG. 5. Deletion of the intron of the tRNATrp gene of H. volcanii does not affect growth in rich medium under standard conditions. H. volcanii strains H26 and H26Wi were grown in 25 ml of Hv-YPC medium (1) in sidearm flasks at 42°C in triplicate experiments. Growth was monitored with a Klett Summerson colorimeter. Mean values with standard deviations are plotted. Results of a repeat experiment showed similar values.

Conclusions. Gene disruptions have been used to characterize the in vivo activity of snoRNAs in eukaryotes (18). This is the first report of the deletion of a specific sRNA-containing intron sequence from the genome of an archaeon and its effect in vivo. Previously we showed that sRNA present in the intron of tRNA^Trp of H. volcanii could guide 2'-O methylation of C34 and U39 of pre-tRNA^Trp in vitro (25). The present in vivo study confirmed this function of the sRNA. The absence of intron-dependent sRNA-guided modifications of an essential RNA product of a single-copy gene did not affect the cell viability. Since this guide sRNA-containing intron was retained in the tRNA^Trp gene during haloarchaeal and some other euryarchaeal evolution, the effect of intron deletion may manifest itself under certain stress or competitive conditions.

 
We thank Thorsten Allers for providing H. volcanii strain H26 and the pTA131 plasmid and David Clark for critical review of the manuscript.

This work was supported by NIH grant GM55045 to R.G.

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Southern Illinois University, 1245 Lincoln Drive, Carbondale, IL 62901-4413. Phone: (618) 453-6466. Fax: (618) 453-6440. E-mail: rgupta{at}siumed.edu

Published ahead of print on 29 August 2008.

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Journal of Bacteriology, November 2008, p. 7308-7313, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00820-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Joardar, A. Articles by Gupta, R. Search for Related Content PubMed PubMed Citation Articles by Joardar, A. Articles by Gupta, R.