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Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.

Nakayama H, Akiyama M, Taoka M, Yamauchi Y, Nobe Y, Ishikawa H, Takahashi N, Isobe T - Nucleic Acids Res. (2009)

Bottom Line: Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values.The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts.It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
We present here a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. Ariadne, a unique web-based database search engine, identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts. It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

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Nucleotide sequences of the mature tRNAPhe-1 (Mature tRNA) and the equivalent transcript identified in the yeast tRNA database (Database). The identified RNase T1 fragments are underlined in the upper half of the figure and are listed in the lower half. Lower case letters indicate the methylated forms of the corresponding unmodified nucleotides (e.g. g for methylated G), and ‘D’ indicates dihydrouridine. Other symbols for modified nucleotides are according to Limbach et al. (47): ‘L’, 2-methylguanosine; ‘D’, dihydrouridine; ‘R’, N2,N2-dimethylguanosine; ‘B’, 2′-O-methylcytidine; ‘#’, 2′-O-methylguanosine; ‘Y’, wybutosine; ‘P’, pseudouridine; ‘?’, 5-methylcytidine; ‘7’, 7-methylguanosine; ‘T’, 5-methyluridine; and ‘"’, 1-methyladenosine. The boxed sequence indicates an intron.
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Figure 5: Nucleotide sequences of the mature tRNAPhe-1 (Mature tRNA) and the equivalent transcript identified in the yeast tRNA database (Database). The identified RNase T1 fragments are underlined in the upper half of the figure and are listed in the lower half. Lower case letters indicate the methylated forms of the corresponding unmodified nucleotides (e.g. g for methylated G), and ‘D’ indicates dihydrouridine. Other symbols for modified nucleotides are according to Limbach et al. (47): ‘L’, 2-methylguanosine; ‘D’, dihydrouridine; ‘R’, N2,N2-dimethylguanosine; ‘B’, 2′-O-methylcytidine; ‘#’, 2′-O-methylguanosine; ‘Y’, wybutosine; ‘P’, pseudouridine; ‘?’, 5-methylcytidine; ‘7’, 7-methylguanosine; ‘T’, 5-methyluridine; and ‘"’, 1-methyladenosine. The boxed sequence indicates an intron.

Mentions: To evaluate whether Ariadne can be used to identify post-transcriptional modifications in RNA, we analyzed the RNase T1 digest of tRNAPhe-1 (Supplementary Figure S3), and the MS/MS data were used to search against a Saccharomyces cerevisiae tRNA database transcribed from tDNA sequences [The Genomic∼tRNA Database, http://lowelab.ucsc.edu/GtRNAdb/(40)] under a search condition that considers up to two modifications and permits up to two missed cleavages. After carrying out automated two-step evaluations of the MS/MS data against all entries in the database, Ariadne clearly identified tRNAPhe-1 with seven oligonucleotides and assigned five of the seven oligonucleotides with the methylated nucleotides and/or dihydrouridines (Figure 5). In this application, Ariadne discriminated tRNAPhe-1 from its structural homologue tRNAPhe-2 by the fragment AAUUCG, which is unique to tRNAPhe-1; however, more detailed analysis indicated that the tRNAPhe-1 preparation used in this study contained small amount of tRNAPhe-2 (Taoka et al., manuscript in preparation). Figure 6 illustrates the identification of monomethylated adenosine (mA) in the sequence 5′-OH-mAUCCACAG-cyclic phosphate-3′ (indicated by an arrow in Supplementary Figure S3). In fact, we could identify all monomethylated nucleotides in the yeast tRNAPhe-1 by the automated LC–MS/MS–Ariadne analysis, except for the hypermodified oligonucleotide containing yW and the intron sequence.Figure 5.


Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.

Nakayama H, Akiyama M, Taoka M, Yamauchi Y, Nobe Y, Ishikawa H, Takahashi N, Isobe T - Nucleic Acids Res. (2009)

Nucleotide sequences of the mature tRNAPhe-1 (Mature tRNA) and the equivalent transcript identified in the yeast tRNA database (Database). The identified RNase T1 fragments are underlined in the upper half of the figure and are listed in the lower half. Lower case letters indicate the methylated forms of the corresponding unmodified nucleotides (e.g. g for methylated G), and ‘D’ indicates dihydrouridine. Other symbols for modified nucleotides are according to Limbach et al. (47): ‘L’, 2-methylguanosine; ‘D’, dihydrouridine; ‘R’, N2,N2-dimethylguanosine; ‘B’, 2′-O-methylcytidine; ‘#’, 2′-O-methylguanosine; ‘Y’, wybutosine; ‘P’, pseudouridine; ‘?’, 5-methylcytidine; ‘7’, 7-methylguanosine; ‘T’, 5-methyluridine; and ‘"’, 1-methyladenosine. The boxed sequence indicates an intron.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Nucleotide sequences of the mature tRNAPhe-1 (Mature tRNA) and the equivalent transcript identified in the yeast tRNA database (Database). The identified RNase T1 fragments are underlined in the upper half of the figure and are listed in the lower half. Lower case letters indicate the methylated forms of the corresponding unmodified nucleotides (e.g. g for methylated G), and ‘D’ indicates dihydrouridine. Other symbols for modified nucleotides are according to Limbach et al. (47): ‘L’, 2-methylguanosine; ‘D’, dihydrouridine; ‘R’, N2,N2-dimethylguanosine; ‘B’, 2′-O-methylcytidine; ‘#’, 2′-O-methylguanosine; ‘Y’, wybutosine; ‘P’, pseudouridine; ‘?’, 5-methylcytidine; ‘7’, 7-methylguanosine; ‘T’, 5-methyluridine; and ‘"’, 1-methyladenosine. The boxed sequence indicates an intron.
Mentions: To evaluate whether Ariadne can be used to identify post-transcriptional modifications in RNA, we analyzed the RNase T1 digest of tRNAPhe-1 (Supplementary Figure S3), and the MS/MS data were used to search against a Saccharomyces cerevisiae tRNA database transcribed from tDNA sequences [The Genomic∼tRNA Database, http://lowelab.ucsc.edu/GtRNAdb/(40)] under a search condition that considers up to two modifications and permits up to two missed cleavages. After carrying out automated two-step evaluations of the MS/MS data against all entries in the database, Ariadne clearly identified tRNAPhe-1 with seven oligonucleotides and assigned five of the seven oligonucleotides with the methylated nucleotides and/or dihydrouridines (Figure 5). In this application, Ariadne discriminated tRNAPhe-1 from its structural homologue tRNAPhe-2 by the fragment AAUUCG, which is unique to tRNAPhe-1; however, more detailed analysis indicated that the tRNAPhe-1 preparation used in this study contained small amount of tRNAPhe-2 (Taoka et al., manuscript in preparation). Figure 6 illustrates the identification of monomethylated adenosine (mA) in the sequence 5′-OH-mAUCCACAG-cyclic phosphate-3′ (indicated by an arrow in Supplementary Figure S3). In fact, we could identify all monomethylated nucleotides in the yeast tRNAPhe-1 by the automated LC–MS/MS–Ariadne analysis, except for the hypermodified oligonucleotide containing yW and the intron sequence.Figure 5.

Bottom Line: Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values.The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts.It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
We present here a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. Ariadne, a unique web-based database search engine, identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts. It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

Show MeSH