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A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA

A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA
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  REPORT A novel partial modification at C2501in  Escherichia coli   23S ribosomal RNA THOMAS EMIL ANDERSEN, 1 BO TORBEN PORSE, 2 and FINN KIRPEKAR 1 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark 2 Section for Gene Therapy Research, Copenhagen University Hospital, Juliane Mariesvej 20, DK-2100 Copenhagen, Denmark ABSTRACT Escherichia coli   is the best-characterized organism with respect to posttranscriptional modifications of its ribosomal RNA(rRNA). It is presently believed that all the modified nucleotides have been identified, primarily on the basis of two detectionmethods; modification-induced inhibition of the enzyme reverse transcriptase or analysis by combined HPLC and electrosprayionization mass spectrometry. Comparison of data from these different approaches reveals a disagreement regarding modifi-cation of C2501 in  E. coli   23S rRNA. A. Bakin and J. Ofengand previously reported the detection of a modification at this sitebased on a reverse transcriptase assay. J.A. McCloskey and coworkers could not confirm the existence of such a modificationusing an electrospray ionization mass spectrometry approach. C2501 is therefore generally considered unmodified. We haveused a strategy involving isolation of a specific rRNA fragment from  E. coli   23S rRNA followed by Matrix Assisted LaserDesorption/Ionization mass spectrometry and tandem mass spectrometry to investigate this controversy. Our data reveal anovel 16-Da partial modification at C2501. We believe that the data reported here clarify the above discrepancy, because aminor partial modification detected in a reverse transcriptase assay would not necessarily be detected by the srcinal massspectrometry approach. The level of modification was furthermore monitored in different growth situations, and we found asignificant positive regulation in stationary phase cells. C2501 is universally conserved and implicated in structure folds veryclose to the catalytic center of the ribosome. Moreover, several antibiotics bind to nucleotides in this region, which altogethermake a modification at this site interesting.Keywords:  E. coli   rRNA modification; growth phase dependent; MALDI mass spectrometry; tandem mass spectrometry INTRODUCTION Maturation of stable RNA in the cell generally involves ahigh degree of modification during and following genera-tion of the primary transcript. The type of modificationstaking place at the single nucleotide level is termed post-transcriptional modifications. These are especially frequentin tRNA, but also appear in rRNA, and typically involveaddition of small chemical groups that change the physicaland chemical properties of the nucleotide.  Escherichia coli 16S and 23S rRNA are modified at 11 and 23 residues,respectively (Rozenski et al. 2000), and the major part of themodifications are found on highly conserved nucleotides inor near the functional centers of the ribosome (Bakin andOfengand 1993; Brimacombe et al. 1993). Several studieshave shown posttranscriptional modifications to be indis-pensable for normal ribosomal assembly, translational ac-tivity, and fidelity of decoding (e.g., Cunningham et al.1991; Green and Noller 1996; O’Connor et al. 1997; Caldaset al. 2000), but details in the function of posttranscription-ally modified nucleotides are poorly understood.The contemporary methods used to detect and charac-terize modified nucleotides in RNA are reverse transcriptasemapping (e.g., Bakin and Ofengand 1993) and analysis by acombination of HPLC and electrospray ionization massspectrometry (ESI-MS; Kowalak et al. 1993). HPLC/ESI-MSenables parallel measurement of precise molecular mass andchromatographic mobility, and has therefore proven effec-tive and reliable, not only to verify reverse transcriptase databut also for detection and mapping of novel modifications.The initial step in the LC/MS approach is a total nucleosideanalysis to quantify and identify modified nucleosides. Spe-cific chemical modification or tandem mass spectrometry may furthermore be used. Localization in the primary se-quence is established by LC/MS on defined oligoribo-nucleotides produced enzymatically by digestion with theguanosine-specific RNase T1. A subsequent comparison of  Reprint requests to:  Finn Kirpekar, Department of Biochemistry andMolecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; e-mail: and publication are at RNA  (2004), 10:907–913. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 RNA Society.  907  the measured masses with those expected from rDNA datareveals fragments harboring modified nucleotides.We have previously demonstrated the use of Matrix As-sisted Laser Desorption/Ionization (MALDI) mass spec-trometry and tandem mass spectrometry as alternativemethods to analyze RNA modifications (Kirpekar et al.2000; Kirpekar and Krogh 2001). The MALDI technique isgenerally advantageous in terms of sensitivity, toleranceagainst impurities, and ability to handle complex mixtures.The latter is a result of a predominant generation of singly charged molecular ions, leading to dramatically reducedcomplexity of the spectrum. Samples containing even a highnumber of analyte molecules, such as RNase digestions of large RNAs, are therefore typically resolved into a numberof distinct peaks, each representing a single analyte species.The properties of MALDI MS therefore make preanalyticseparation steps, such as HPLC, less necessary. MALDI tan-dem mass spectrometry offers the above-mentioned quali-ties of the MALDI spectrum and, in addition, enables pre-cursor ion selection and ion fragmentation. It is a means of sequencing and of detailed structural analysis of shortstretches of RNA.In the present work, MALDI MS and MALDI tandem MSwere used to detect and localize a previously unknownmodification in domain V—the peptidyltransferase cen-ter—of   E. coli  23S rRNA. We were encouraged to investi-gate this region by unpublished data from a previous study,which indicated occurrence of a partial 16-Da modificationnear position 2500 (B.T. Porse and F. Kirpekar, unpubl.). Areverse transcriptase assay published in 1993 by Bakin andOfengand furthermore indicated an unknown modificationat C2501. This suggested modification was later investigatedby HPLC/ESI-MS analysis with a negative outcome (Ko-walak et al. 1995). No link has afterward been establishedbetween the reverse transcription data and a modified resi-due. Here, we resolve this apparent conflict by showing thata fraction of C2501 in  E. coli  23S rRNA is indeed subjectedto modification. This was first detected as a 16-Da massincrement of the RNase T1 fragment containing nucleotides2496-C-A-Cm-C-U-C-G-2502. The modification was sub-sequently localized to C2501 by a combination of tandemmass spectrometry of the above 7-mer and parallel massspectrometric analysis of RNase A digests of the purifiedrRNA segment C2480–C2527. RESULTS AND DISCUSSIONDetection of a partial modificationin the RNase T1 fragment C2496–G2502 A successful MALDI MS analysis of rRNA strongly dependson correctly prepared and purified samples. The prelimi-nary steps of this study therefore included the developmentof a suitable method to site-specifically isolate rRNA frag-ments expected to carry novel modifications. For this pur-pose, we utilized a strategy based on nuclease protection of an rRNA sequence by hybridization to a complementary DNA oligodeoxynucleotide (Maden 1980). This concept hasalso been adapted for use with mass spectrometry (Kowalak et al. 1995). However, we found it necessary to optimize theprocedure with respect to hybridization and digestion. Inparticular, we found hybridization to be more efficient us-ing a HEPES/KCl annealing buffer in a slow-cool proce-dure, and the addition of a small amount of RNase A to-gether with the single-strand-specific mung bean nucleasepromoted proper digestion. The changes were found to im-prove yield significantly, and ultimately enabled us to usethe method on starting material down to  ∼ 10 pmoles.Because we wished to isolate a short segment covering thenucleotides around position 2500 of   E. coli  23S rRNA, aDNA oligonucleotide complementary to the region C2480–C2527 was hybridized to  E. coli  total rRNA. Single-strandedDNA and RNA were subsequently removed by nucleasedigestion, and the protected RNA fragment was gel-puri-fied. This RNA fragment was then digested to completionby the guanine-specific RNase T1 and directly analyzed by MALDI time-of-flight (TOF) mass spectrometry (Kirpekaret al. 2000). Figure 1A displays a typical mass spectrumfrom an MALDI MS analysis of the RNase T1 digest. Com-parison between the expected fragment masses listed in Fig-ure 1B and those in the spectrum immediately confirms theidentity of the sequence C2480–C2527. The high accuracy of mass determination (better than one-tenth of a dalton)as well as a good signal-to-noise ratio additionally allowsthe detection of a peak at  m / z   = 2249.27. An enlargement of the  m / z   = 2190–2290 region (Fig. 1A, inset) shows an iso-topic distribution as expected from an RNA fragment of this mass. The presence of this unexpected peak is inter-preted as a novel 16-Da partial modification of a nucleotidein the digestion fragment 2496-C-A-Cm-C-U-C-G-2502.The  m / z   = 2249.3 peak was detected repeatedly in severalother preparations, including preparations from a differentstrain of   E. coli . In all samples, similar relative intensities of the  m / z   2249.3 and 2233.3 peaks were observed, showingthe fraction of 16-Da modified 23S rRNA to be 10%–30%.The low frequency of modification is not due to thermaldegradation during the 90°C denaturation step (see Mate-rials and Methods), as significant variations in the times of 90°C treatment did not influence the observed ratio (datanot shown). We can also rule out that the 2249.3-Da frag-ment is an artifact from the nuclease isolation procedure,because the same modified digestion fragment was observedin a previous work where the initial  ∼ 50-mer rRNA fragmentwas purified by means of oligodeoxynucleotide annealing andRNase H digestion (B.T. Porse and F. Kirpekar, unpubl.). Localization of the modified nucleotide To narrow down the possible sites of modification, we em-ployed tandem mass spectrometry using a MALDI qua- Andersen et al. 908  RNA, Vol. 10, No. 6  drupole-TOF mass spectrometer (Kirpekar and Krogh2001) to sequence the +16-Da-modified 7-mer. Favorably,two different phosphate forms are often produced by RNaseT1 digestion; one with the normal 3  -phosphate and onewith its 2  -3  -cyclic precursor (Kirpekar et al. 2000). Tan-dem mass spectrometry of the 7-mer in both phosphateforms as well as their 16-Da heavier posttranscriptionally modified counterparts generated two sets of comparabledata. Supported by tandem mass spectrometry data fromthe normal 3  -phosphate species, peaks were assigned in thetandem mass spectra of the 2215.35-Da 2  -3  -cyclic phos-phate and the +16-Da-modified species as shown in Figure 2.Essentially all major peaks are accounted for, and the pres-ence of many backbone cleavages facilitates a partial se-quencing of the two RNAs. Comparison of the spectra re-veals that only some ions appear at equal  m / z   ratios,whereas the remaining ions display a characteristic 16-Damass shift to a higher value. This makes it possible to nar-row down nucleotides likely to be modified, by excludingnucleotides residing in fragment ions present at the same m / z   value in the two spectra.The nucleotides 2496-C-A-Cm-C-2499 at the 5  -end of the oligoribonucleotide are immediately ruled out, as the5  -sequence ions containing some or all of these nucleo-tides, that is, a 3 , c 3 , d 3 , a 4 , appear at thesame  m / z   ratios in the two spectra (a, b,c, and d ions are backbone cleavage ionscontaining the srcinal 5  -end, whereasw, x, y, and z ions contain the srcinal3  -end; nomenclature according toMcLuckey et al. 1992). This interpreta-tion is supported by the peaks corre-sponding to the w  3 , y  3 , w  4 , y  5 , and y  6 ions, which all retain a 16-Da incrementin the tandem mass spectrum of themodified species. The exclusion of posi-tions 2496-C-A-Cm-C-2499 as puta-tive sites of a 16-Da modification wasfully substantiated by tandem massspectrometry analyses of the modi-fied and unmodified 3  -phosphatespecies (data not shown). Minor dis-crepancies between the spectra, as wellas the absence of some importantsequence ions, unfortunately impeded FIGURE 1.  Fragment C2480–C2527 of   E. coli  23S rRNA digested with RNase T1. (  A ) MALDI time-of-flight mass spectrum of the  m / z   regioncovering trinucleotides or larger. The  inset   reveals the +16.0-Da partial modification of the 2496-CACmCUCG-2502 fragment at  m / z   2249.27. ( B )A list of the expected RNase T1 digestion fragments based on the rRNA sequence with presently known nucleotide modifications. m 2 A is2-methyladenosine, and  indicates pseudouridine. FIGURE 2.  Tandem mass spectra of the 2496-CACmCUCG-2502 fragment and its 16-Da-larger modified derivative (2215.35 and 2231.26 Da ions; 2  -3  -cyclic phosphate versions of the m / z   2233.32 and 2249.27 ions shown in Fig. 1A). Lowercase letters refer to backbone cleavagefragments, where a, b, c, and d contain the srcinal 5  -end, and w, x, y, and z the srcinal3  -end. Associated numbers are the length of the fragment in nucleotides. Capital letters referto the nucleotides of internal fragments or, if preceded by (-) to loss of the indicated nucleo-base. Modification of C2501 in   E. coli  23S rRNA  909  further analysis of the 3  -end of the7-mer, and unambiguous assign-ment of the 16-Da modification to asingle of the 3  -proximal nucleotides2500-U-C-G-2502 was therefore notpossible based on tandem mass spec-trometry.To determine the exact site of modi-fication, U2500, C2501, and G2502were examined by mass spectro-metric analysis of the C2480–C2527fragment digested with the pyrimidinespecific RNase A. This digestion re-leases U2500 and C2501 as mono-nucleotides, whereas G2502 resides inthe trinucleotide 2502-G-m 2 A-  -2504.A modified G2502 would be revealedby a partial mass shift of this latter frag-ment, as G2502 is the only nucleo-tide common to the RNase T1 diges-tion fragment in which the partialmodification was initially detected. No2502-G-m 2 A-  -2504 +16-Da frag-ment was observed (data not shown),which therefore excludes G2502 asbeing modified. The  m / z   region cover-ing single nucleotides is shown for theRNase A digest in Figure 3A. It re-veals three nonmatrix peaks at  m / z  324.05, 325.05, and 340.09, corre-sponding to cytidine monophos-phate, uridine monophosphate, and aputative +16-Da-modified cytidinemonophosphate. The  m / z   340.1 peak was repeatedly detected in independ-ent rRNA preparations. We carefully analyzed RNA-free, but otherwise iden-tical, samples to identify signals srci-nating from the matrix, the buffer, andso on (Fig. 3B). Subsequently, we de-phosphorylated the RNase A digest toconfirm the identity of the  m / z   340.1signal as a modified cytidine mono-phosphate. As shown in Figure 3C,the modified nucleoside as well as Cand U nucleosides appear at  m / z   values80 Da lower than in Figure 3A upondephosphorylation. Comparison withspectra of RNA-free matrix controlsshowed that these peaks were specificto the RNase-digested samples (datanot shown). This unambiguously dem-onstrates that the +16-Da posttran-scriptional modification resides onC2501.  FIGURE 3.  (Legend on next page) Andersen et al. 910  RNA, Vol. 10, No. 6  Putative roles of the 16-Da modification at C2501 In case of a physiological role, a regulatory dependencewould be a likely reason for the partial nature of the modi-fication. Modulation of the rRNA modification level hasbeen suggested as a means to cope with extreme growthtemperatures (e.g., Noon et al. 1998). We tested this corre-lation for the C2501 modification, but observed no signifi-cant temperature dependence (data not shown). Anotherfactor known to affect the level of nucleotide modificationin RNA is the growth phase at isolation (e.g., Singhal andVold 1976). We therefore analyzed rRNA extracted fromcells harvested at three different time points in the station-ary phase. In this case, our measurements showed a signifi-cant up-regulation of the 16-Da modification upon transi-tion from exponential to stationary growth (Fig. 4). Themodification level in stationary cells was consistently foundto be 40%–60 %, that is, a rise in modification level of   ∼ 2×compared with exponentially growing cells. We cannot ruleout the possibility of a more pronounced regulation in re-sponse to other environmental factors, maybe leading tocomplete modification. Further knowledge of the modifi-cation such as identity and pathway of synthesis is needed tomake any biological suggestions from these observations.Nevertheless, the dependence on growth phase reveals aninteresting and previously unseen dynamic behavior of nucleotide modifications in  E. coli  rRNA.It is difficult to judge whether the modification at C2501is purposely synthesized at this position, or if the modifi-cation is a side effect created by, for example, a tRNA-modifying enzyme. Indications for the latter would be itsrelatively low abundance and the fact that 16-Da cytidinemodifications have, as yet, only been detected in tRNA, notin ribosomal RNA. One explanation for the modificationmay therefore involve recognition of the motif aroundC2501 by a tRNA-modifying enzyme. However, some as-pects of the 16-Da modification, such as its spatial localiza-tion and its growth phase dependence, make it worth con-sidering possible structural/functional roles in the local ri-bosomal environment. C2501 is implicated in maintain-ing the tertiary structure near the catalytic center, becauseit forms a conserved wobble pair with A2450, positioningit within 6 Å of the proposed active-site residue A2451(Ban et al. 2000; Nissen et al. 2000). A role of the modifi-cation at C2501 could be to stabilize this H-bonding, andthereby function in the maintenance of local spatial struc-tures.C2501 is furthermore localized in a hot spot for bindingof peptidyl transferase antibiotics, in particular the strepto-gramin A and B drugs (for review, see Porse et al. 2000).Using a UV-cross-linking procedure, the therapeutically important streptogramin B antibiotic pristinamycin IA waspreviously found to bind tightly at position U2500/C2501 inauthentic 23S rRNA (Porse et al. 1999). Interestingly, natu-ral, posttranscriptionally modified rRNA was required fordrug binding, as no cross-link was observed using full-length T7 23S rRNA transcripts. This was interpreted toindicate involvement of one or more posttranscriptionalmodifications in the sequence 2498-Cm-C-U-C-G-m 2 A-  -G-2505. Our finding that C2501 is, indeed, partially modi-fied itself, suggests that the 16-Da modification at C2501may, directly or indirectly, be implicated in pristinamycinIA binding. Alternatively, the modification at C2501 may simply render this nucleotide a more potent UV-cross-link-ing reactant. Conclusion By application of the sensitive MALDI MS technique, weinvestigated the functionally important peptidyltransferasecenter of   E. coli  23S rRNA for the pres-ence of unknown modification. Ownunpublished results, as well as otherpublished data, suggested the existenceof a modification at or near C2501,making this site the focus of the presentstudy. By combining a preparationmethod based on nuclease protectionwith mass spectrometric and tandemmass spectrometric analysis, it was pos- FIGURE 4.  The  m / z   region 2190–2290 from an MALDI time-of-flightmass spectrum of the C2480–C2527 fragment of   E. coli  23S rRNAdigested with RNase T1. rRNA in this sample was isolated from sta-tionary-phase cells. The 2496-CACmCUCG-2502 fragment at  m / z  2233.34 and its 16-Da-larger derivative at 2249.31 appear at aboutequal intensities. FIGURE 3.  MALDI time-of-flight mass spectra of the region covering single nucleotides. (  A )Fragment C2480–C2527 of   E. coli  23S rRNA digested with the pyrimidine specific RNase A.Single cytidine and uridine monophosphates produced appear at the  m / z   values 324.05 and325.05, respectively (the relative low intensity of the uridine monophosphate ion is caused by its lower proton affinity; Liguori et al. 2000). A peak corresponding to a 16-Da-modifiedcytidine monophosphate is furthermore observed at  m / z   340.09. ( B ) The peaks of analyte ionsare close to, but do not overlap, seven distinct matrix peaks in this region shown in theRNA-free control. ( C  ) MALDI time-of-flight mass spectrum of a sample prepared as in  A , butwith an additional treatment with alkaline phosphatase. The peaks corresponding to cytidine,uridine, and 16-Da-modified cytidine are shifted to an 80-Da-lower value, corresponding toloss of phosphate. Modification of C2501 in   E. coli  23S rRNA  911
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