RNase mapping of intact nucleic acids by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and 18O labeling

Meng, Zhaojing; Limbach, Patrick A.

doi:10.1016/j.ijms.2004.01.004

Cited by 26 publications

(23 citation statements)

References 38 publications

(58 reference statements)

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“…Liquid chromatography-mass spectrometry (LC-MS)/MS RNase mapping and sequencing of nucleic acids has been used extensively for the localization of modified nucleosides in RNAs (17)(18)(19)(20)(21). Here, we used separate digestions of tRNA Ile 2 with RNase T1 and RNase A combined with bacterial alkaline phosphatase (BAP) followed by LC-MS/ MS to confirm C34 as the site of the unknown modification and to identify the mass of the modified nucleoside.…”

Section: Resultsmentioning

confidence: 99%

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Mandal

Köhrer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Modification of the cytidine in the first anticodon position of the AUA decoding tRNA Ile (tRNA Ile 2 ) of bacteria and archaea is essential for this tRNA to read the isoleucine codon AUA and to differentiate between AUA and the methionine codon AUG. To identify the modified cytidine in archaea, we have purified this tRNA species from Haloarcula marismortui, established its codon reading properties, used liquid chromatography-mass spectrometry (LC-MS) to map RNase A and T1 digestion products onto the tRNA, and used LC-MS/MS to sequence the oligonucleotides in RNase A digests. These analyses revealed that the modification of cytidine in the anticodon of tRNA Ile 2 adds 112 mass units to its molecular mass and makes the glycosidic bond unusually labile during mass spectral analyses. Accurate mass LC-MS and LC-MS/MS analysis of total nucleoside digests of the tRNA Ile 2 demonstrated the absence in the modified cytidine of the C2-oxo group and its replacement by agmatine (decarboxy-arginine) through a secondary amine linkage. We propose the name agmatidine, abbreviation C þ , for this modified cytidine. Agmatidine is also present in Methanococcus maripaludis tRNA Ile 2 and in Sulfolobus solfataricus total tRNA, indicating its probable occurrence in the AUA decoding tRNA Ile of euryarchaea and crenarchaea. The identification of agmatidine shows that bacteria and archaea have developed very similar strategies for reading the isoleucine codon AUA while discriminating against the methionine codon AUG.agmatine | decoding | RNA modification | tRNA | wobble pairing T he genetic code table consists of sixteen four-codon boxes. In fourteen of the boxes, all four codons either specify the same amino acid or are split into two sets of two codons, with each set encoding a different amino acid. For example, the UUN box is split into UUU/UUC coding for phenylalanine and UUA/UUG coding for leucine. The wobble hypothesis of Crick proposes how a single phenylalanine tRNA with G in the first anticodon position can base pair with either U or C and a single leucine tRNA with a modified U (or 2-thioU) in the anticodon can base pair with either A or G (1-3). The two remaining boxes, UGN and AUN, are exceptions in that the UGN box is split into UGU/UGC coding for cysteine, UGG coding for tryptophan, and UGA being used as a stop codon, whereas the AUN box is split into AUU/AUC/AUA coding for isoleucine and AUG coding for methionine. The isoleucine codons AUU and AUC can be read by an isoleucine tRNA with G in the anticodon following the wobble pairing rules, but how the AUA codon is read specifically by a tRNA Ile without also reading the AUG codon has been a question of much interest over the years.Different organisms have developed different strategies for reading the AUA codon. Bacteria use a tRNA Ile with the anticodon LAU (L ¼ lysidine) (4-7). Lysidine is a modified cytidine in which the C2-oxo group of cytidine is replaced by lysine. Exactly how it base pairs with A but not with G is not established.Eukaryotes, on the other hand, con...

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Section: Resultsmentioning

confidence: 99%

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Mandal

Köhrer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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111

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“…This challenge is particularly noteworthy for larger digestion products wherein the "all light" (C-12) isotope peak is no longer the most abundant. Because we have shown that digestion with MC1 ensures a single uridine will be present at the 5 ′ -terminus of each digestion product (with the bulky modification exception as noted above), the number of cytidines should also be more easily determined based on accurate mass measurements Meng and Limbach 2004), and prior sequence reconstruction challenges will be minimized.…”

Section: Discussionmentioning

confidence: 99%

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 fromMomordica charantia

Addepalli

Lesner

Limbach

2015

RNA

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A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA Tyr I . Analysis of MC1 digestion products by ion-pairing reverse phase liquidchromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5 ′ -termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.

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“…Importantly, the presence of other small RNAs, such as 5S rRNA, does not complicate or interfere with the detection of tRNA signature digestion products, and a single MALDI analysis is sufficient to identify these tRNAs. The ribonucleasedigested transfer RNAs were also labeled with 18 O and analyzed separately by MALDI-MS (Meng and Limbach 2004) to confirm the expected peaks (data not shown). All of the major peaks in the mass spectra are easily assignable to expected digestion products, and all expected products generate distinct signals.…”

Section: Analysis Of E Coli Cell Lysatementioning

confidence: 99%

“…Characterization of RNAs by MS is commonly done at two levels: oligonucleotide analysis through the selective digestion of intact RNAs with endonucleases, and/or sequencing of oligonucleotides through the use of tandem mass spectrometry via collision-induced dissociation or post-source decay (Limbach 1996;Nordhoff et al 1996). We and others have shown that RNAs can be characterized by mass spectrometry through the selective digestion of intact RNAs with endonucleases, which can be analyzed using liquid chromatography electrospray ionization mass spectrometry (Kowalak et al 1993(Kowalak et al , 2000McCloskey et al 2001) or MALDI-MS (Polo and Limbach 1998;Kirpekar et al 2000;Berhane and Limbach 2003a,b;Hartmer et al 2003;Meng and Limbach 2004). While these RNase mapping approaches have been used previously to identify post-transcriptionally modified RNAs, here we develop a more extensive RNA identification strategy based upon the generation of unique or signature RNase digestion products.…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products

Hossain¹,

Limbach²

2006

RNA

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The separation of biologically active, pure, and specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA identity and/or abundance are difficult. We have discovered that the enzymatic digestion of an individual tRNA by a ribonuclease (e.g., RNase T1) will generate digestion products unique to that particular tRNA, and we show that a comparison of an organism's complete complement of tRNA RNase digestion products yields a set of unique or ''signature'' digestion product(s) that ultimately enable the detection of individual tRNAs from a total tRNA pool. Detection is facilitated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and proof-of-principle is demonstrated on the whole tRNA pool from Escherichia coli. This method will enable the individual identification of tRNA isoacceptors without requiring specific affinity purification or extensive chromatographic and/or electrophoretic purification. Further, experimental identifications of tRNAs or other RNAs will now be possible using this signature digestion product approach in a manner similar to peptide mass fingerprinting used in proteomics, allowing RNomic studies of RNA at the post-transcriptional level.

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RNase mapping of intact nucleic acids by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and 18O labeling

Cited by 26 publications

References 38 publications

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 fromMomordica charantia

Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products

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RNase mapping of intact nucleic acids by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and 18O labeling

Cited by 26 publications

References 38 publications

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA Ile , base pairs with adenosine but not with guanosine

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA Ile , base pairs with adenosine but not with guanosine

Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 fromMomordica charantia

Mass spectrometry-based detection of transfer RNAs by their signature endonuclease digestion products

Contact Info

Product

Resources

About

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine

Agmatidine, a modified cytidine in the anticodon of archaeal tRNA ^Ile , base pairs with adenosine but not with guanosine