Exoribonuclease R in Mycoplasma genitalium can carry out both RNA processing and degradative functions and is sensitive to RNA ribose methylation

Abstract

Mycoplasma genitalium, a small bacterium having minimal genome size, has only one identified exoribonuclease, RNase R (MgR). We have purified MgR to homogeneity, and compared its RNA degradative properties to those of its Escherichia coli homologs RNase R (EcR) and RNase II (EcII). MgR is active on a number of substrates including oligoribonucleotides, poly(A), rRNA, and precursors to tRNA. Unlike EcR, which degrades rRNA and pre-tRNA without formation of intermediate products, MgR appears sensitive to certain RNA structural features and forms specific products from these stable RNA substrates. The 3'-ends of two MgR degradation products of 23S rRNA were mapped by RT-PCR to positions 2499 and 2553, each being 1 nucleotide downstream of a 2'-O-methylation site. The sensitivity of MgR to ribose methylation is further demonstrated by the degradation patterns of 16S rRNA and a synthetic methylated oligoribonucleotide. Remarkably, MgR removes the 3'-trailer sequence from a pre-tRNA, generating product with the mature 3'-end more efficiently than EcII does. In contrast, EcR degrades this pre-tRNA without the formation of specific products. Our results suggest that MgR shares some properties of both EcR and EcII and can carry out a broad range of RNA processing and degradative functions.

FIGURE 1.

Purification of recombinant M. genitalium RNase R (MgR) from E. coli. About 5 μg of protein from each fraction were denatured, separated on a 4%–15% SDS-polyacrylamide gel, and stained with Coomassie blue. (lane 1) Crude cell extract from culture of Rosetta-gami (DE3)/pLysS harboring pETmgR before IPTG induction; (lane 2) crude extract from culture after IPTG induction; (lane 3) pooled Affi-gel Blue (AGB) peak fractions; (lane 4) flowthrough of hydroxyapatite (HT) column; (lane 5) peak fractions of Sephadex S200 column. In addition, E. coli RNase R (EcR) was purified as described previously (Cheng and Deutscher 2002; Zuo et al. 2006) and is shown in lane 6. The migration positions of molecular mass standards are shown on the left (in kilodaltons). The positions of MgR and EcR are indicated by arrows on the right.

FIGURE 2.

Degradation of polyadenylates by M. genitalium RNase R. RNase R activity on [H³]-poly(A) substrate was determined by acid soluble assay as described in Materials and Methods. Specific activities [in nanomoles of poly(A) per minute per milligram of enzyme] under various conditions are plotted as mean±standard error from three independent experiments. (A) pH range with 10 μM ZnCl₂ and 100 mM KCl; (B) ZnCl₂ or MgCl₂, with 100 mM KCl at pH 8.5; (C) KCl, with 10 μM ZnCl₂ at pH 8.5.

FIGURE 3.

Degradation of oligoribonucleotides. Oligonucleotides were labeled at their 5′-ends with ³²P. Assays were carried out at 37°C in 40 μL reaction mixtures containing 25 μM of the indicated substrate; 1.0 μg of enzyme (EcR or MgR) was used in these analyses. Lanes labeled as “Buffer” indicate control reactions with no enzymes added. Five-microliter aliquots were taken at the times indicated, and the reactions were stopped with 2 volumes of RNA loading buffer. Products were resolved on 22.5% denaturing polyacrylamide gels. The sequences of the oligonucleotides used in this experiment are as follows: C4, CA14 (5′-CCCCACCACCAACA-3′), and C17.

FIGURE 4.

Degradation of E. coli rRNA by MgR and EcR. Isolated rRNA was treated with RNases in Zn²⁺-containing buffer for the length of time indicated on the top of each lane. Agarose gels (1.2%) were used to separate the RNA. A photograph was taken under UV lights after staining with SYBR Gold. Intermediate 23S RNA degradation products are labeled on the right side by arrows. The RNA products were quantified using the integrated optical density of the bands detected by the Epi Chemi II Darkroom (UVP Laboratory Products). Gels from three independent runs were quantified, and average values with standard errors are shown in the bottom panels. (A) Reactions with equal amounts of MgR and EcR (0.15 μg each). (B) Reactions with MgR and EcR of similar poly(A) degradation activity (0.5 μg of MgR or 0.038 μg of EcR).

FIGURE 5.

Determination of the 3′-end of the 23S and 16S rRNA degradation products by MgR. RNA was purified by phenol extraction from reactions with buffer, MgR (RNA from reactions in Fig. 4B, lanes 3,9), or EcR (reactions in Fig. 4A, lane 6, and Fig. 4B, lane 7). Linker was ligated to RNA of both samples. RT-PCR was carried out as described in Materials and Methods. PCR products were separated on agarose gel. (A) RT-PCR products from 23S rRNA. (Lane 1) RT-PCR from RNA treated with buffer using primer pair P1 (RP + 23S-LP2). A product with an expected size of 769 bp from full-length 23S RNA was formed. (Lane 2) RT-PCR using the same RNA and primer pair P2 (RP + 23S-LP1). An expected product of 553 bp from full-length 23S RNA is shown. (Lanes 4,5) RT-PCR products from RNA treated with MgR using P1 and P2, respectively. Estimated sizes of the products are indicated on the right side of the gel. (Lanes 3,6) Fifty-base pair DNA Step Ladder (Promega). (B) RT-PCR products from 16S rRNA. cDNA samples were prepared using RNA from reactions indicated on the top of each lane. RT-PCR was carried out using either primer pair P3 (RP + 16S-LP1, producing 354-bp DNA from full-length 16S rRNA) or primer pair P4 (RP + 16S-LP2, producing 294-bp DNA from full-length 16S rRNA). (Lane 1) Fifty-base pair DNA Step Ladder (Promega). (Lanes 2,3) RT-PCR from RNA treated with buffer (Fig. 4B, lane 3), showing the expected products of (lane 2) 354 bp and (lane 3) 294 bp from full-length 16S rRNA. In addition, less abundant products of (lane 2) ∼220 bp and (lane 3) ∼160 bp were also detected. (Lanes 4,5) RT-PCR from RNA treated with 500 ng of MgR for 60 min (Fig. 4B, lane 9). (Lanes 6,7) RT-PCR from RNA treated with 150 ng of EcR for 30 min (Fig. 4A, lane 6). (Lanes 8,9) RT-PCR from RNA treated with 37.5 ng of EcR for 120 min (Fig. 4B, lane 7). (C) RNA secondary structure representation of the 3′-region of E. coli 23S rRNA (adapted from Cannone et al. 2002a, with permission from BioMed Central, ©2000; kindly made available by Dr. Robin Gutell's group at http://www.rna.icmb.utexas.edu/). (Arrows) MgR stop sites; (numbers, boxes, and lines) various tertiary interactions between the numbered nucleotides.

FIGURE 6.

Degradation of a 2′-O-methylated oligoribonucleotide by MgR, EcR, and EcII. Reactions were carried out and products were separated using the same condition as in Figure 3. The sequence of this 17-mer oligoribonucleotide is 5′-CAGUUG(Um)GAUCGAUCCC-3′. The labeled 17-mer was treated with buffer or RNase in a time course indicated on the top of the gel. A [³²P]-labeled RNA Decade Markers (Ambion) was included in the gel (not shown), and the sizes are shown on the left. The sizes of RNA products are shown on the right.

FIGURE 7.

3′-RACE of M. genitalium tRNA₁ ^Gly products. RNA was ligated to the DNA linker as described in Materials and Methods and in the legend of Figure 5. RT-PCR was carried out using primer pairs P1 (RP + tRNA-LP1), P2 (RP + tRNA-LP2), P3 (RP + tRNA-LP3), or a single primer RP. The expected PCR products from the mature 3′-end of tRNA₁ ^Gly using P1, P2, or P3 are labeled as M1, M2, or M3, respectively. A 25-bp DNA Step Ladder (25bp ladder; Promega) was used as the size marker in each gel. (A) RT-PCR products from total RNA isolated from M. genitalium culture (M.g. RNA). (B) PCR products using DNA templates gel-isolated from A: (Lane 2) DNA1 products above M1, up to ∼100 bp longer than M1; (lane 3) DNA2 products above M2, up to ∼100 bp longer than M2. Major PCR products longer than the products from mature tRNA are labeled Pre1, Pre2, and Pre3 on the right. (C) RT-PCR products from the 74-nt RNA species generated from treatment of pre-tRNA₁ ^Gly by MgR. Total RNA from M. genitalium (M.g. RNA) was used to show the sizes of PCR products from mature tRNA₁ ^Gly.

FIGURE 8.

Processing and degradation of M. genitalium tRNA₁ ^Gly precursor. Pre-tRNA₁ ^Gly with a mature 5′-end and a 21-nt 3′-trailer was uniformly labeled with ³²P and was treated with RNases as described in Materials and Methods. RNA products were detected by autoradiography after separation on an 8% denaturing polyacrylamide gel. (Lane 1) A [³²P]-labeled RNA ladder (RNA Decade Markers; Ambion) was used as the size marker. (Lanes 2,3) In addition, 78-nt and 74-nt RNA transcripts containing the same sequence as tRNA₁ ^Gly were used as size markers. These markers were generated using PCR templates from the tRNA gene, starting from the 5′-end of the tRNA and ending at the 3′-end (for 74 nt) or 4 nt downstream (for 78 nt). Pre-tRNA₁ ^Gly was treated by buffer (containing 10 μM ZnCl₂), EcR, MgR, or EcII for 30 min at 37°C in the presence of various concentrations of MgCl₂ as indicated on the top of each lane. (P) The size of the precursor is 95 nt. (M) The size of mature tRNA₁ ^Gly is 74 nt.