Box C/D RNA-guided 2'-O methylations and the intron of tRNATrp are not essential for the viability of Haloferax volcanii

Abstract

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

FIG. 1.

Primary sequence and predicted secondary structure of the H. volcanii pre-tRNA^Trp and tRNA^Trp. (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 . (B) Mature tRNA^Trp (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.

FIG. 2.

The tRNA^Trp gene of H. volcanii strain H26ΔWi does not contain the intron. (A) PCR products using H26 and H26ΔWi 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 H26ΔWi 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 H26ΔWi with exon-specific primers was sequenced. The arrow denotes the junction between the two exons, indicating precise deletion of the intron (see Fig. 1B).

FIG. 3.

The H. volcanii strain H26ΔWi genome does not contain intron of tRNA^Trp gene. The figure shows PhosphorImager analyses of Southern blots of SalI-digested DNA of H. volcanii strains H26 (lanes 1 and 3) and H26ΔWi (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.

FIG. 4.

Intron-dependent sRNA-guided 2′-O-methylated residues are not present in the tRNA^Trp of H. volcanii strain H26ΔWi. (A) Thin-layer chromatography of RNase T2 (left panels) and nuclease P1 (right panels) digests of the tRNA^Trp of strains H26 and H26ΔWi. 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 tRNA^Trp of strains H26 and H26ΔWi 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 tRNA^Trp samples are represented in lanes 1 and 2. The “bands” corresponding to U29, U33, and U39 are labeled.

FIG. 5.

Deletion of the intron of the tRNA^Trp gene of H. volcanii does not affect growth in rich medium under standard conditions. H. volcanii strains H26 and H26ΔWi 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.