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RNA. May 2006; 12(5): 725–733.
PMCID: PMC1440906

Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution

Abstract

Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution and therefore were present in the last common ancestor. In this paper we report that KsgA orthologs from archaeabacteria and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life.

Keywords: dimethyltransferase, KsgA, Dim1, rRNA modification, ribosome biogenesis

INTRODUCTION

Ribosome biogenesis is a fundamental cellular process, and its importance is underscored by the effort required to assemble functional ribosomes. Although ribosome maturation proceeds by different pathways in different organisms, it always requires precise coordination of a complicated set of steps including numerous cleavages and modifications of rRNA, assembly of the ribosomal proteins onto the rRNA, and involvement of a variety of other factors of unknown function. Functional eubacterial ribosomes can be assembled in vitro using only the component rRNAs and ribosomal proteins (Held et al. 1973), although additional factors are required in vivo for optimal assembly (Lövgren and Wikström 2001; Culver 2003). Eukaryotic ribosome assembly is more complex, involving a large number of extra ribosomal factors (Venema and Tollervey 1999; Warner 2001). In addition, ribosome assembly in eukaryotes is spatially and temporally organized, with early processing and assembly steps taking place in the nucleolus and the final steps being completed after transport to the cytoplasm (Venema and Tollervey 1999; Schafer et al. 2003). Both prokaryotic and eukaryotic ribosomes contain numerous modifications in their RNA, including pseudouridylation, base methylation, and ribose methylation. While eubacteria require separate enzymes for most modifications, eukaryotic cells generally utilize a common enzyme for each type of modification, using snoRNAs to guide the specific modifications (Fromont-Racine et al. 2003).

There is a dearth of information on ribosome assembly in archaeal systems. Archaeal rRNA modification seems to be more similar to that found in eukaryotic organisms, with snoRNAs guiding pseudouridylation and ribose methylation (Omer et al. 2003). Indeed, guide RNAs from archaeal organisms can direct methylation of Xenopus laevis RNA (Speckmann et al. 2002). Although archaeal cells lack a nuclear membrane, at least one archaeal genome contains putative homologs to nuclear and nucleolar structural genes from eukaryotes (Pace 1997). Spatio-temporal control of archaeal ribosome synthesis is therefore a possibility, although speculative.

While post-transcriptional modification of rRNA is common to all life, in most cases it is not very well characterized or understood. In some cases, dispensing with the modification produces no phenotype, while knockout of the enzyme has a deleterious effect, indicating that these proteins have other unknown functions that are essential to the proper functioning of the cell (Sirum-Connolly and Mason 1993; Gutgsell et al. 2000, 2001; Ofengand et al. 2001). Although all organisms contain rRNA modifications, very few of these have been conserved throughout evolution. A notable exception is the dimethylation of two adjacent adenosines in the 3′-terminal helix of small subunit rRNA (Helix 45 in Escherichia coli). Helix 45 is one of the most highly conserved sequences in small subunit rRNA (van Knippenberg et al. 1984; Mears et al. 2002), and the presence of two dimethylated adenosines in the loop of the helix is equally conserved. The only known exceptions are the small rRNAs of Euglena gracilis chloroplasts and Sulfolobus solfataricus, each of which contain only a single dimethylated adenosine (van Buul et al. 1984; Noon et al. 1998), and Saccharomyces cerevisiae mitochondria, which lack dimethyladenosines altogether (Klootwijk et al. 1975). The dimethylated adenosine residues are thus a rare example of an evolutionarily conserved post-transcriptional rRNA modification.

The enzyme responsible for dimethylation of these two adenosines is also universally conserved; in all known instances it has been found to be a member of the KsgA/Dim1 family of methyltransferases. The enzyme was first described in E. coli (Helser et al. 1972; Poldermans et al. 1979) and has since been described in eubacteria (van Buul et al. 1983) and eukaryotes (Lafontaine et al. 1994; Housen et al. 1997), and in chloroplasts (Tokuhisa et al. 1998) and mitochondria (Seidel-Rogol et al. 2003). While the enzyme has not yet been described in archaeabacteria, putative KsgA orthologs can be found in the genomes of these organisms.

KsgA is able to methylate fully formed 30S subunits, although these subunits must be in a translationally inactive conformation to support methylation (P. Desai and J. Rife, unpubl.). However, the true in vivo substrate for KsgA is unknown. A minimal in vitro substrate has been found to consist of 16S rRNA plus a subset of ribosomal proteins (Thammana and Held 1974). This suggests that KsgA methylates a discrete intermediate in the 30S assembly pathway. Orthologs from yeast and from human mitochondria have been shown to complement for KsgA function in bacteria (Lafontaine et al. 1994; Seidel-Rogol et al. 2003). This indicates that the bacterial, eukaryotic, and organellar enzymes can recognize a common bacterial substrate, despite differences in the respective 30S maturation processes.

The KsgA family of proteins has been exploited evolutionarily; as cellular organization and ribosome biogenesis grew more complex, KsgA orthologs were recruited to play additional roles within the cell. The yeast version of KsgA, which is known as Dim1, is essential for proper processing of the primary rRNA transcript (Lafontaine et al. 1995). Depletion of Dim1 leads to buildup of aberrant rRNA species. Another KsgA ortholog, Pfc1, has been identified in the chloroplasts of Arabidopsis thaliana (Tokuhisa et al. 1998). This enzyme is the methyltransferase for chloroplast small subunit rRNA and is also important for proper chloroplast development at low temperatures. h-mtTFB, which is a recently described KsgA ortholog in human mitochondria, serves as a mitochondrial transcription factor as well as being the dimethyltransferase for mitochondrial 12S rRNA (McCulloch et al. 2002; Seidel-Rogol et al. 2003). h-mtTFB also has an intriguing link with human disease, having been shown to be a phenotypic modifier of deafness associated with a polymorphic A1555G mutation in mitochondrial rRNA (Bykhovskaya et al. 2004). In the cases of both Dim1 and h-mtTFB, the methylating activity is separate from the second function; methylation-deficient mutations can be made, which leave the rRNA processing or transcription activity intact (Lafontaine et al. 1998; McCulloch and Shadel 2003).

In this article we report for the first time the cloning and characterization of an archaeal KsgA family member, which we term MjDim1, from Methanocaldococcus jannaschii. MjDim1 activity is described both in vivo and in vitro in bacterial systems. We also examine the activity of Dim1 from S. cerevisiae, confirming in vivo activity in bacteria and demonstrating its activity in vitro. For clarity, this protein will be referred to as ScDim1. Our results indicate that the core methyltransferase activity of this family of proteins, including recognition of a complex substrate, has evolved little since the last common ancestor.

RESULTS AND DISCUSSION

Cloning, expression, and purification of proteins

To identify the archaeal ortholog of the KsgA/Dim1 family we used NCBI's genomic BLAST tool to search the M. jannaschii sequence, using the E. coli KsgA protein sequence (accession no. P06992) as the query sequence. This search identified a putative ortholog, accession number NP_248023, which was annotated as dimethyladenosine transferase (ksgA) in the Entrez protein record. Alignment of this protein with other KsgA/Dim1 family members revealed a high degree of similarity (Fig. (Fig.1),1), including the presence of motifs common to SAM-dependent methyltransferases. We followed the eukaryotic nomenclature and designated the protein as MjDim1 (for M. jannaschii dimethyltransferase 1).

We obtained M. jannaschii genomic DNA from ATCC and amplified the gene encoding MjDim1 by PCR, using primers designed from the coding sequence (accession no. NC_000909). The gene was cloned into the pET15b vector, in-frame with the vector-encoded N-terminal His tag, and was confirmed by sequencing. The plasmid was transformed into BL-21 (DE3) cells for protein production.

The identification and cloning of ScDim1 was described by Lafontaine et al. (1994) and was provided to us by the same group as a pET15b construct engineered to contain an N-terminal histidine tag. Analysis of the ScDim1 gene sequence revealed the use of codons that are rare in E. coli; therefore, the protein was expressed in BL21-CodonPlus (DE3)-RIL cells, which contain extra copies of rare codons. Protein purification was made difficult by insolubility of the overexpressed protein; expression conditions were optimized to produce the majority of protein in a soluble form.

KsgA was cloned as previously described (O'Farrell et al. 2003) and transformed into BL-21 (DE3) cells. All proteins were overexpressed with IPTG and purified to homogeneity by Ni2+ affinity chromatography.

In vivo analysis

ScDim1 has been shown to complement for KsgA function in ksgA E. coli cells (Lafontaine et al. 1994), demonstrating functional equivalence of the two proteins. We asked whether MjDim1 could also complement KsgA knockout. In vivo activities of both ScDim1 and MjDim1 were assessed using a modified minimal inhibitory concentration (MIC) assay, which takes advantage of the fact that KsgA knockout renders bacteria resistant to the antibiotic kasygamycin (ksg) (Helser et al. 1972; van Buul et al. 1983). Plasmids containing the two proteins were transformed into a ksgR strain of E. coli, which lacks endogenous KsgA activity. This strain was constructed from BL-21 (DE3) cells; this allows leaky expression from the pET15 T7 promoter. Growth on ksg was compared to cells transformed with KsgA plasmid (positive control) and cells transformed with empty vector (negative control). Unlike in a traditional MIC, untransformed cells are naturally resistant to the antibiotic and become sensitive when transformed with a functional dimethylase. Therefore, cells transformed with KsgA have a low MIC of 400 μg/mL ksg, while cells transformed with an empty vector have a high MIC, >3000 μg/mL ksg. As shown in Table Table1,1, MjDim1 is fully functional in this in vivo system, with an MIC of 400 μg/mL. ScDim1, on the other hand, shows partial activity on bacterial ribosomes in vivo, with an MIC of 1200 μg/mL. While ScDim1 does not restore full sensitivity to the antibiotic, it does show increased sensitivity, indicating that the enzyme is able to recognize the small subunit as a substrate. Lack of full complementation may correlate with slower and/or incomplete methylation of 30S as compared to the other two enzymes (see nucleotide analysis).

TABLE 1
In vivo activity assay

In vitro analysis

We next asked how efficiently ScDim1 and MjDim1 were able to methylate E. coli 30S in an in vitro assay. Unmethylated 30S subunits were prepared from the ksgR strain described above. Incorporation of 3H-methyl from labeled SAM by each enzyme was followed at discrete time points over an interval of 2 h. Control experiments were performed with 30S subunits purified from wild-type E. coli cells, which are methylated by endogenous KsgA and thus do not serve as substrates. Initial experiments were performed with 10 pmol of 30S substrate and 1 pmol of enzyme. This amount of protein did not allow for completion of the reaction within 2 h, so experiments were also performed using 10 pmol each of 30S and enzyme.

Figure Figure2,2, A–C, shows the time course of methylation for KsgA, ScDim1, and MjDim1, respectively. Methylation of E. coli 30S by ScDim1 and MjDim1 closely followed the KsgA time course, both in rate of incorporation and final level of methylation. In reactions with 1 pmol of protein, MjDim1 showed a slightly higher rate of 3H incorporation than KsgA and ScDim1 at later time points. With stoichiometric amounts of protein, the time course of methylation was essentially indistinguishable between the three proteins, thus confirming the ability of the enzymes from archaebacteria and eukaryotes to recognize bacterial 30S subunits as substrates.

FIGURE 2.
In vitro methylation of 30S. Time-course assays for KsgA (A), ScDim1 (B), and MjDim1 (C). Diamonds indicate assays containing 10 pmol ksgR 30S, 10 pmol enzyme; squares indicate assays containing 10 pmol ksgR 30S, 1 pmol enzyme; triangles indicate ...

We then estimated the amount of methyl groups transferred at the 2-h time point by each enzyme, with both 1 pmol and 10 pmol amounts of enzyme (Fig. (Fig.2D),2D), by constructing a standard curve of cpm versus concentration of 3H-methyl-SAM. With 10 pmol of 30S per reaction, and four methylation sites per 30S molecule, we would expect to see transfer of 40 pmol methyl groups if the reactions have gone to completion. As shown in Figure Figure2D,2D, our calculations lead to slight overestimation of methyl group transfer, probably due to error in 3H counting. However, the reactions performed with 10 pmol enzyme appear to be more or less complete after 2 h. Reactions performed with only 1 pmol enzyme are approximately halfway completed after 2 h.

Nucleoside analysis

In vitro assays of the three proteins, performed as described above for the time course, were incubated for 2 h and analyzed to determine relative amounts of m6A and m6 2A (Fig. (Fig.2E;2E; Table Table2).2). 16S rRNA isolated from 30S subunits methylated by 10 pmol of either KsgA or MjDim1 contained no detectable labeled m6A; radioactive incorporation was seen only in the dimethyladenosine peak. This agrees with the in vitro data suggesting that these reactions have gone to completion (Fig. (Fig.2D).2D). ScDim1, on the other hand, produced a mixture of m6A and m6 2A; ~28% of the incorporated radiolabel was found on monomethylated adenosine. This indicates that at most 80% of the potential sites were methylated after 2 h.

TABLE 2
Quantitation of methylated adenosine species

Partially methylated 30S from reactions using 1 pmol of enzyme showed radioactive peaks at both m6A and m6 2A. Surprisingly, although the total level of methylation was similar for all three enzymes (Fig. (Fig.2D),2D), rRNA methylated by the different enzymes showed different ratios of m6A to m6 2A. ScDim1 produced ~1.4 times as much m6A as m6 2A. MjDim1, conversely, produced almost no m6A; only ~1% of the incorporated methyl groups were found on m6A. KsgA fell somewhere between the other two, producing both m6A and m6 2A, with only 0.8 times as much m6A as m6 2A. These results could be a result of assaying enzymes from different species on the bacterial substrate, or they could reflect a difference in reaction mechanism.

The KsgA/Dim1 enzymes transfer a total of four methyl groups from four SAM molecules to two adenosines. The exact mechanism of transfer has not yet been established; questions remain as to order of addition, if any, and the number of binding events required for the four methylations. There is some evidence that, under stringent conditions that limit the SAM concentration to produce only 50% methylation, KsgA preferentially dimethylates the 3′-proximal adenosine, suggesting a possible sequence of methylation steps (van Buul et al. 1984). However, mutation of either adenosine still allows dimethylation of the other, indicating that methylation order, if it exists, is only preferred and not obligatory (Cunningham et al. 1990). There are no data concerning released intermediates, or how many methyl groups are transferred per binding event.

The above data begin to address the question of the multiple methyl group transfers. Partially methylated 30S were produced in reactions containing 10 pmol 30S subunits and 1 pmol enzyme. Therefore, m6A produced in excess of 2 pmol (corresponding to two adenosines available for methylation per subunit) will only be seen if the enzyme releases the substrate after monomethylation and rebinds to a new substrate. With 20.1 pmol of methyl groups incorporated by 1 pmol ScDim1, the 1.4:1 m6A:m6 2A ratio represents ~8.3 pmol labeled m6A and 5.9 pmol labeled m6 2A (1 pmol m6 2A represents 2 pmol incorporated methyl groups). Therefore, under our assay conditions, ScDim1 clearly releases the m6A intermediate, which is subsequently converted to the m6 2A product after an additional binding event.

In contrast, there is no indication that MjDim1 produces m6A as anything but a transient intermediate. Of the 24.1 pmol methyl groups transferred, only 0.3 pmol were found on m6A. These results suggest that the archaeal enzyme preferentially forms dimethyladenosine, without release of the monomethyl intermediate. While release of a monomethyl intermediate and subsequent rebinding and addition of the second methyl cannot be ruled out, such a model requires that MjDim1 prefer the monomethylated substrate to the unmethylated substrate to a large degree.

In terms of m6A versus m6 2A production, KsgA falls somewhere in between ScDim1 and MjDim1. Unlike ScDim1, KsgA produces less m6A than m6 2A; however, of the 18.1 pmol of methyl groups transferred, 5.2 pmol are found on m6A, which is still indicative of a released intermediate. Although it is possible that these differences are a result of suboptimal assay conditions, these results also allow the possibility of distinct mechanisms for the three enzymes, thus demonstrating a need for future analysis to dissect the exact scheme of methyl transfer.

Conclusions

Substrate recognition by the KsgA/Dim1 methyltransferases is complex. KsgA is able to methylate 30S subunits under conditions of low Mg++, but it can also methylate a pre-ribosomal particle containing 16S and a partial complement of ribosomal proteins (Thammana and Held 1974). Dim1 is essential for early processing of the pre-23S rRNA, but does not methylate 23S until very late in the 40S maturation process (Lafontaine et al. 1995). Despite evolutionary divergence of ribosomal assembly and processing pathways, eukaryotic and archaeal KsgA orthologs are able to methylate E. coli 30S both in vivo and in vitro. This requires the conservation of similar structural cues in small ribosomal subunits across evolution. Also complex is the mechanism of the modification performed by these enzymes. A total of four methyl groups are transferred, from four SAM molecules, to two separate adenosines. It is clear from the crystal structure of KsgA (O'Farrell et al. 2004) that only one SAM molecule is bound at a time, and that the adenosines enter the active site separately. It has not been determined in what order, if any, the methyl groups are transferred, or if all four of the transfers take place within a single or multiple binding events. The work presented here demonstrates clear differences in the reaction intermediate profiles produced in vitro by KsgA enzymes from bacteria, archaea, and yeast, despite the fact that the three enzymes methylate bacterial 30S to a similar extent and at similar rates in the assay used. However, we cannot exclude the possibility that the differences in the respective rates of m6A and m6 2A production seen here are a result of suboptimal substrate rather than a reflection of true differences in mechanism. For example, the yeast and archaeal enzymes may show different activity if assayed on their respective 30S subunits rather than on bacterial 30S. Our results demonstrate the remarkable cross-recognition of a complex substrate by evolutionarily distant members of an enzyme family and emphasize the need to further investigate the multistep reaction mechanism.

MATERIALS AND METHODS

Cloning of MjDim1

M. jannaschii genomic DNA was obtained from ATCC. The gene encoding MjDim1 was amplified with the following primers, purchased from Integrated DNA Technologies: 5′-GCCGCACCATATGTTCAAACCAAAGAAAAAATTAGG-3′ and 5′-GCTACTCGAGCTATAACCTACCCCTATTTTGCAG-3′. The amplicon was then inserted into pET15b as an NdeI–XhoI fragment. The correct clone was confirmed by sequencing (Nucleic Acids Research Facilities, Virginia Commonwealth University).

Protein expression and purification

pET15b–KsgA and pET15b–MjDim1 plasmids were transformed into BL-21 (DE3) cells for overexpression. Cell cultures were grown to an OD600 of 0.6 in the presence of ampicillin and induced with 1 mM IPTG (Sigma-Aldrich). After 4 h at 37°C, cells were harvested by centrifugation. Pellets were resuspended in lysis buffer (50 mM NaPO4, 300 mMNaCl, 10 mM imidazole at pH 8.0), broken with two passages through an Emulsiflex cell breaker (Avestin), and centrifuged to remove cell debris. Cleared lysate was loaded onto a HiTrap Chelating column (Amersham) equilibrated with 0.1 M NiSO4, washed twice with increasing amounts of imidazole (wash buffer 1: 50 mM NaPO4, 300 mM NaCl, 20 mM imidazole at pH 8.0; wash buffer 2: 50 mM NaPO4, 300 mM NaCl, 50 mM imidazole at pH 8.0), and the protein eluted with elution buffer (50 mM NaPO4, 300 mM NaCl, 250 mM imidazole at pH 8.0).

The pET15b–ScDim1 construct was provided by Dr. Jean Vandehaute and was confirmed by sequencing. Protein was expressed in BL21-CodonPlus (DE3)-RIL cells (Stratagene). The cells were grown at 37°C to an OD600 of 1.2 in the presence of ampicillin. Then the protein was induced under mild conditions with 0.1 mM IPTG and transferred to 25°C for 4 h. Cells were harvested and broken as for KsgA and MjDim1. Purification was carried out by affinity chromatography using a Ni2+ column; buffers included 15% glycerol and 3 mM 2-mercaptoethanol along with the above concentrations of NaH2PO4, NaCl, and imidazole. To increase the stability of the protein, glycerol and 2-mercaptoethanol were added to the purified protein to final concentrations of 25% and 6 mM, respectively.

Proteins were estimated to be >95% pure by SDS-PAGE analysis. Protein concentration was measured using the Bio-Rad Protein Assay.

30S purification

An E. coli strain lacking functional KsgA was constructed by growing BL-21 (DE3) cells on ksg to select for loss of the dimethylations. 30S ribosomes from this ksgR strain were used in an in vitro assay to confirm that the adenosines were able to be methylated, and therefore that the resistance to ksg was due to lack of KsgA activity. 30S subunits were prepared as previously described (Blaha et al. 2000), except that cells were broken as described above. Purified subunits were dialyzed into reaction buffer (40 mM Tris at pH 7.4; 40 mM NH4Cl; 4 mM MgOAc; 6 mM 2-mercaptoethanol) and stored at –80°C in single-use aliquots. 30S concentration was estimated by measuring the absorbance at 260 nm and using a relationship of 67 pmol 30S per 1 unit of optical density.

In vivo assay

ksgR cells were transformed with the pET15b constructs and selected on LB plates containing ampicillin. Transformed colonies were picked into liquid media and grown in overnight culture. These cultures were diluted 1:25 in fresh LB containing 50 μg/mL ampicillin and grown to OD600 of 0.7–0.8, diluted 1:100 in fresh LB, and plated onto LB/ampicillin containing increasing amounts of ksg, from 0 to 3000 μg/mL. Plates were incubated at 37°C overnight and visually inspected for colony formation.

In vitro assay

The in vitro assay was adapted from Poldermans et al. (1979). Time-course reactions were performed in 500 μL volumes containing 40 mM Tris (pH 7.4), 40 mM NH4Cl, 4 mM MgOAc, 6 mM 2-mercaptoethanol, 0.02 mM 3H-methyl-SAM (780 cpm/pmol; MP Biomedicals), 100 pmol 30S subunits, and 10 or 100 pmol enzyme; volume and components were sufficient for 10 reactions. Buffer and reagents were prewarmed to 37°C and added into prewarmed tubes to minimize any lag in the reaction start. At each of eight designated time points 50 μL was removed and added to a prechilled tube containing 10 μL of 100 mM unlabeled SAM (Sigma-Aldrich) to quench the reaction; the remaining 100 μL was stored at −20°C and used for HPLC analysis (see below). The quenched reactions were deposited onto DE81 filter paper (Whatman), washed twice with ice-cold 5% TCA, and rinsed briefly with ethanol. Filters were air-dried for 1 h, placed into scintillation fluid, and counted.

HPLC analysis

Labeled 16S rRNA was extracted from 30S subunits with phenol/chloroform/isoamyl alcohol. 16S was digested and dephosphorylated as described by Gehrke and Kuo (1989), and subjected to nucleoside analysis by reversed-phase HPLC. Nuclease P1 was obtained from USBiological, shrimp alkaline phosphatase was from MBI Fermentas. HPLC analysis was performed on a Polaris C-18 column (Varian). The HPLC system used consisted of a Waters 600 Controller, a Waters 2487 Dual λ Absorbance Detector, and Waters Empower software. Radioactivity was monitored with a Packard 150TR Flow Scintillation Analyzer. Buffer A was 20 mM NaH2PO4 (pH 5.1). Buffer B was 20 mM NaH2PO4 (pH 5.1):acetonitrile 70:30. Separation was performed at room temperature using a linear gradient from 100%A-100%B over 20 min, at a flow rate of 1.0 mL/min. Nucleoside standards used were N6-methyladenosine (Sigma-Aldrich) and N6 N6-dimethyladenosine, synthesized as in Rife et al. (1998). Peak integration was calculated by the Empower software and used to determine ratios of m6A:m6 2A.

ACKNOWLEDGMENTS

We thank Dr. Jean Vandenhaute for kindly providing us with the pET15b-Dim1 construct. This work was supported by grants to J.P.R. from the Jeffress Memorial Trust, the A. D. Williams Foundation, and the NIH (GM66900).

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2310406.

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