#METABOLOMICS WORKBENCH atsai_20250207_210246 DATATRACK_ID:5605 STUDY_ID:ST003728 ANALYSIS_ID:AN006115 PROJECT_ID:PR002314
VERSION             	1
CREATED_ON             	February 13, 2025, 5:23 pm
#PROJECT
PR:PROJECT_TITLE                 	Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals
PR:PROJECT_TITLE                 	a m7G in the Conserved P-loop of 23S rRNA
PR:PROJECT_SUMMARY               	Extremophilic archaea employ diverse chemical RNA modifications, providing a
PR:PROJECT_SUMMARY               	rich source of new enzymes for biotechnologically valuable RNA manipulations.
PR:PROJECT_SUMMARY               	Our understanding of the modified nucleoside profiles in Archaea, as well as the
PR:PROJECT_SUMMARY               	functions and dynamic regulation of specific RNA modifications is far from
PR:PROJECT_SUMMARY               	complete. Here, we established an extensive profile of nucleoside modifications
PR:PROJECT_SUMMARY               	in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS
PR:PROJECT_SUMMARY               	analysis and rigorous non-coding RNA depletion, identifying - with high
PR:PROJECT_SUMMARY               	confidence - at least four previously unannotated modifications in archaeal
PR:PROJECT_SUMMARY               	mRNAs. Nucleoside quantification analysis conducted on total, large, small, and
PR:PROJECT_SUMMARY               	mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus
PR:PROJECT_SUMMARY               	kodakarensis revealed a series of modifications whose abundance is dynamically
PR:PROJECT_SUMMARY               	responsive to growth temperatures, implying that specific RNA modifications are
PR:PROJECT_SUMMARY               	fitness relevant under specific growth conditions. To predict the RNA-modifying
PR:PROJECT_SUMMARY               	enzymes most likely to generate the new and dynamic RNA modifications, we
PR:PROJECT_SUMMARY               	leveraged a bioinformatics analysis of open-access databases to annotate likely
PR:PROJECT_SUMMARY               	functional domains of archaeal proteins. Putative enzyme activities were
PR:PROJECT_SUMMARY               	confirmed in vitro and in vivo by assessing the presence of the target RNA
PR:PROJECT_SUMMARY               	modification in genetic deletion strains of T. kodakarensis. Our approach led to
PR:PROJECT_SUMMARY               	the discovery of a methyltransferase-encoded gene responsible for m7G
PR:PROJECT_SUMMARY               	modification in the P-loop of 23S rRNA peptidyl transferase center and validates
PR:PROJECT_SUMMARY               	a novel and effective platform for discovering RNA-modifying enzymes through
PR:PROJECT_SUMMARY               	LC-MS/MS analysis that will accelerate efforts of the community towards
PR:PROJECT_SUMMARY               	uncovering the complex and dynamic roles of RNA modifications.
PR:INSTITUTE                     	New England Biolabs
PR:LAST_NAME                     	Tsai
PR:FIRST_NAME                    	Yueh-Lin
PR:ADDRESS                       	44 Dunham Ridge, Beverly, Massachusetts, 01915, USA
PR:EMAIL                         	atsai@neb.com
PR:PHONE                         	978-380-6587
#STUDY
ST:STUDY_TITLE                   	Identification and quantification of a m7G modification in T. kodakarensis
ST:STUDY_TITLE                   	ribosomal RNA
ST:STUDY_SUMMARY                 	To investigate the activity of the protein encoded by TK0008, we conducted a
ST:STUDY_SUMMARY                 	comprehensive nucleoside analysis comparing the total RNA from the TS559
ST:STUDY_SUMMARY                 	wild-type strain with that of the TK0008-deletion strain.
ST:INSTITUTE                     	New England Biolabs
ST:LAST_NAME                     	Tsai
ST:FIRST_NAME                    	Yueh-Lin
ST:ADDRESS                       	44 Dunham Ridge, Beverly, Massachusetts, 01915, USA
ST:EMAIL                         	atsai@neb.com
ST:PHONE                         	978-380-6587
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Thermococcus kodakarensis
SU:TAXONOMY_ID                   	311400
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	TS559_rep1-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_01-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_rep1-r002	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_01-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_rep1-40x	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_40x_total_RNAmods_01.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_rep1-r001	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_02-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_rep1-r002	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_total_RNAmods_02-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_rep1-40x	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=Tk_40x_total_RNAmods_02.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_rep2-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_01.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_rep2-20x	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_20x_01.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_rep2-r001	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_02.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_rep2-20x	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Total_20x_02.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_total_RNA_rep1-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_Expo_TotalRNA_2018-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_total_RNA_rep1-r002	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_Expo_TotalRNA_2018-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_total_RNA_rep1-r001	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Expo_TotalRNA_2018-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_total_RNA_rep1-r002	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_Expo_TotalRNA_2018-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_total_RNA_rep2-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_01-1-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_total_RNA_rep2-r002	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_01-1-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_total_RNA_rep2-r001	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_02-1-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_total_RNA_rep2-r002	Genotype:TK0008 deletion | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TK0008_total_m7G_quant_02-1-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_rep1-r001	Genotype:Wild-type | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TS559_23S-Enriched-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_rep1-r002	Genotype:Wild-type | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TS559_23S-Enriched-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_23S_rep1-r001	Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S-Enriched-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_23S_rep1-r002	Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S-Enriched-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_rep2-r001	Genotype:Wild-type | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_01-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_rep2-r002	Genotype:Wild-type | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_01-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_23S_rep2-r001	Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_02-r001.d
SUBJECT_SAMPLE_FACTORS           	-	TK0008_23S_rep2-r002	Genotype:TK0008 deletion | Sample source:23S_rRNA_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TK0008_23S_G_quant_02-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_NE_rep1-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_NE_rep1-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.1uM_rep1-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.1uM_rep1-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.2uM_rep1-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.2uM_rep1-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_1uM_rep1-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_1uM_rep1-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_4uM_rep1-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_4uM_rep1-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_NE_rep2-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01_rep2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_NE_rep2-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_01_rep2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.1uM_rep2-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02_rep2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.1uM_rep2-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_02_rep2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.2uM_rep2-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03_rep2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_0.2uM_rep2-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_03_rep2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_1uM_rep2-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04_rep2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_1uM_rep2-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_04_rep2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_4uM_rep2-r001	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05_rep2-r001.d
SUBJECT_SAMPLE_FACTORS           	-	IVT23S_rTK0008_4uM_rep2-r002	Genotype:Synthetic 23S_rRNA | Sample source:In vitro transcription	RAW_FILE_NAME(Raw_File_Names)=IVT23S_rTK0008_05_rep2-r002.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_Total_RNA_01	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_total_RNA_rep1-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_Total_RNA_02	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_total_RNA_rep2-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Frag_Enrich_01	Genotype:Wild-type | Sample source:23S_Fragment_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Frag_Enrich_rep1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Frag_Enrich_02	Genotype:Wild-type | Sample source:23S_Fragment_Enrichment	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Frag_Enrich_rep2.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_01_NoDepl-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_01-r001-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_01_NoDepl-r002	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_01-r002-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_02_NoDepl-r001	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_02-r001-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_02_NoDepl-r002	Genotype:Wild-type | Sample source:Total RNA	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_02-r002-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_01_Depl-r001	Genotype:Wild-type | Sample source:23S_Fragment_Depletion	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_03-r001-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_01_Depl-r002	Genotype:Wild-type | Sample source:23S_Fragment_Depletion	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_03-r002-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_02_Depl-r001	Genotype:Wild-type | Sample source:23S_Fragment_Depletion	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_04-r001-1.d
SUBJECT_SAMPLE_FACTORS           	-	TS559_23S_m7G_Fragment_02_Depl-r002	Genotype:Wild-type | Sample source:23S_Fragment_Depletion	RAW_FILE_NAME(Raw_File_Names)=TS559_23S_m7G_Fragment_04-r002-1.d
#COLLECTION
CO:COLLECTION_SUMMARY            	The TK0008 open reading frame (ORF) spans positions 7655 to 8755 on the T.
CO:COLLECTION_SUMMARY            	kodakarensis genome. PCR was used to generate an amplicon including the TK0008
CO:COLLECTION_SUMMARY            	ORF flanked by ~700 bp both up- and down-stream that was cloned into the unique
CO:COLLECTION_SUMMARY            	SwaI site of pTS700 (reference Hileman and Santangelo, PMID: 22701112) 82,
CO:COLLECTION_SUMMARY            	resulting in pCSU-TK0008A. The coding sequence of TK0008 was excised from
CO:COLLECTION_SUMMARY            	pCSU-TK0008A using site-directed QuikChange mutagenesis (Agilent Cat# 200516) to
CO:COLLECTION_SUMMARY            	generate pCSU-TK0008B. All plasmid sequences were confirmed by Sanger
CO:COLLECTION_SUMMARY            	sequencing. pCSU-TK0008B was transformed into T. kodakarensis strain TS559 and
CO:COLLECTION_SUMMARY            	plated onto agmatine-free rich media plates as previously described (reference
CO:COLLECTION_SUMMARY            	PMID: 34752288) 85. Transformants were allowed to grow for 4 days anaerobically
CO:COLLECTION_SUMMARY            	at 85 °C and subsequent colonies were picked into rich liquid media lacking
CO:COLLECTION_SUMMARY            	agmatine and grown overnight. Genomic DNA extracted from 1 mL of cells was used
CO:COLLECTION_SUMMARY            	as a template for PCRs using two sets of primer pairs to determine the site and
CO:COLLECTION_SUMMARY            	directionality of plasmid integration into the genome. One primer from each pair
CO:COLLECTION_SUMMARY            	has homology to the genome with the other having homology to the plasmid to
CO:COLLECTION_SUMMARY            	ensure PCR amplification involves a region flanking TK0008 on the genome.
CO:COLLECTION_SUMMARY            	Colonies confirmed to contain proper integrations at the TK0008 locus were
CO:COLLECTION_SUMMARY            	plated on minimal media containing 1 mM agmatine and 100 µM 6-methyl purine and
CO:COLLECTION_SUMMARY            	grown anaerobically at 85 °C for 2-5 days. Genomic DNA from resultant
CO:COLLECTION_SUMMARY            	colonies was used in diagnostic PCRs to identify strains deleted for TK0008
CO:COLLECTION_SUMMARY            	preliminarily. The deletion of TK0008 was ultimately confirmed by Sanger
CO:COLLECTION_SUMMARY            	sequencing. In vitro transcribed (IVT) 23S rRNA was prepared using a linearized
CO:COLLECTION_SUMMARY            	plasmid template (pBluescript II KS(-)) and HiScribe T7 High Yield RNA Synthesis
CO:COLLECTION_SUMMARY            	Kit (NEB, Cat #E2040S). The synthesized transcripts were cleaned up using
CO:COLLECTION_SUMMARY            	Monarch Spin RNA Cleanup Kit (NEB, Cat #T2050L). To perform in vitro methylation
CO:COLLECTION_SUMMARY            	reactions, IVT 23S rRNA was heat-denatured at 95 C for 30 seconds and cooled to
CO:COLLECTION_SUMMARY            	22 C prior to enzyme treatment. Purified recombinant TK0008 (0.1 – 4 uM) was
CO:COLLECTION_SUMMARY            	incubated with 1 uM of IVT 23S rRNA in 1x methyltransferase buffer (25 mM
CO:COLLECTION_SUMMARY            	Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT) supplemented with 160 uM of
CO:COLLECTION_SUMMARY            	S-adenosyl-methionine (SAM) (NEB, Cat #B9003S) at 85 C for 30 minutes. The
CO:COLLECTION_SUMMARY            	reactions were cleaned up using Monarch Spin RNA Cleanup Kit (NEB, Cat #T2040L).
CO:SAMPLE_TYPE                   	Ribonucleic acid, In Vitro Transcription
#TREATMENT
TR:TREATMENT_SUMMARY             	Harvested archaeal cell pellets were resuspended in 10 mL of TRI reagent
TR:TREATMENT_SUMMARY             	(Molecular Research Center, Inc., Cat #TR118). The resuspended cells were
TR:TREATMENT_SUMMARY             	homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP
TR:TREATMENT_SUMMARY             	Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000
TR:TREATMENT_SUMMARY             	g for 5 minutes to precipitate any cell debris. Supernatants were collected
TR:TREATMENT_SUMMARY             	post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research
TR:TREATMENT_SUMMARY             	Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase
TR:TREATMENT_SUMMARY             	separation. RNA from the aqueous phase was isolated by isopropanol precipitation
TR:TREATMENT_SUMMARY             	and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA
TR:TREATMENT_SUMMARY             	contamination. To further purify the DNase I-treated RNA, an equal volume of
TR:TREATMENT_SUMMARY             	acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific,
TR:TREATMENT_SUMMARY             	Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes
TR:TREATMENT_SUMMARY             	to separate the aqueous phase from the organic phase. The aqueous phase
TR:TREATMENT_SUMMARY             	containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1
TR:TREATMENT_SUMMARY             	volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight.
TR:TREATMENT_SUMMARY             	Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved
TR:TREATMENT_SUMMARY             	in nuclease-free water. To enrich m7G-modified rRNA fragment, 50 ug of total RNA
TR:TREATMENT_SUMMARY             	from T. kodakarensis TS559 wild-type strain was mixed with a biotinylated DNA
TR:TREATMENT_SUMMARY             	probe (1 uM) fully complementary to the target sequence in 10 mM Tris buffer (pH
TR:TREATMENT_SUMMARY             	7.5) and heated to 95 C for 2 minutes followed by a ramp down to 22 C.
TR:TREATMENT_SUMMARY             	Biotinylated DNA probe sequence:
TR:TREATMENT_SUMMARY             	5’-Biotin-ACCTTCGAGGTGTGCGCCAGCAACTGCCACT-3’. The unhybridized RNAs were
TR:TREATMENT_SUMMARY             	digested with 5 uL of RNase 4 (NEB, Cat #M1284L) in 1x NEB buffer r1.1 at 37
TR:TREATMENT_SUMMARY             	C for 1 hour. The digestion was stopped by adding 10 L of human placental
TR:TREATMENT_SUMMARY             	RNase inhibitor (NEB, Cat #M0307S) to the reaction mixture and incubated at room
TR:TREATMENT_SUMMARY             	temperature for 10 minutes. Selective enrichment of the probe-hybridized rRNA
TR:TREATMENT_SUMMARY             	region was performed using streptavidin magnetic beads (NEB, Cat #S1421S).
TR:TREATMENT_SUMMARY             	Briefly, 250 uL of streptavidin magnetic beads were washed twice with equal
TR:TREATMENT_SUMMARY             	volume of low-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 50 mM NaCl) and
TR:TREATMENT_SUMMARY             	resuspended in 50 uL of high-salt buffer (100 mM Tris pH 7.5, 10 mM EDTA, 250 mM
TR:TREATMENT_SUMMARY             	NaCl). The high-salt buffer was removed, and the beads were incubated with the
TR:TREATMENT_SUMMARY             	reaction mixture for 15 minutes at room temperature with occasional agitation.
TR:TREATMENT_SUMMARY             	The bead-captured DNA-RNA hybrid was washed and eluted by heating to 80 C for 5
TR:TREATMENT_SUMMARY             	minutes in 30 uL of nuclease-free water. For m7G-modified rRNA fragment
TR:TREATMENT_SUMMARY             	depletion, the same biotinylated DNA probe (1 uM) described above was mixed with
TR:TREATMENT_SUMMARY             	5 ug of total RNA from TS559 strain in the probe hybridization buffer provided
TR:TREATMENT_SUMMARY             	in NEBNext rRNA Depletion Kit (NEB, Cat #E7850X). The probe-hybridized rRNA
TR:TREATMENT_SUMMARY             	mixture was subjected to rRNA depletion according to the NEBNext rRNA Depletion
TR:TREATMENT_SUMMARY             	Kit protocol. Following the enzymatic treatment, the reactions were cleaned up
TR:TREATMENT_SUMMARY             	using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Total or isolated RNAs were digested to nucleosides at 37°C overnight using a
SP:SAMPLEPREP_SUMMARY            	Nucleoside Digestion Mix (NEB, Cat #M0649S). The digested RNAs were subsequently
SP:SAMPLEPREP_SUMMARY            	injected without prior purification on an Agilent 1290 Infinity II UHPLC
SP:SAMPLEPREP_SUMMARY            	equipped with a G7117 diode array detector and an Agilent 6495C
SP:SAMPLEPREP_SUMMARY            	Triple-Quadrupole Mass Spectrometer operating in positive electrospray
SP:SAMPLEPREP_SUMMARY            	ionization (+ESI) mode.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Solvent A pH is 4.5
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II
CH:COLUMN_NAME                   	Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um)
CH:SOLVENT_A                     	100% water; 10mM ammonium acetate
CH:SOLVENT_B                     	100% methanol
CH:FLOW_GRADIENT                 	1%-40% Solvent B in 26.5 min
CH:FLOW_RATE                     	0.6 mL/min
CH:COLUMN_TEMPERATURE            	30
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6495 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass spectrometric data were acquired using dynamic multiple reaction monitoring
MS:MS_COMMENTS                   	(DMRM) mode. Identification of each nucleoside species was based on the
MS:MS_COMMENTS                   	associated retention time and mass transition in the extracted chromatogram.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Mass Response Ratio
MS_METABOLITE_DATA_START
Samples	TS559_rep1-r001	TS559_rep1-r002	TS559_rep1-40x	TK0008_rep1-r001	TK0008_rep1-r002	TK0008_rep1-40x	TS559_rep2-r001	TS559_rep2-20x	TK0008_rep2-r001	TK0008_rep2-20x
Factors	Genotype:Wild-type | Sample source:Total RNA	Genotype:Wild-type | Sample source:Total RNA	Genotype:Wild-type | Sample source:Total RNA	Genotype:TK0008 deletion | Sample source:Total RNA	Genotype:TK0008 deletion | Sample source:Total RNA	Genotype:TK0008 deletion | Sample source:Total RNA	Genotype:Wild-type | Sample source:Total RNA	Genotype:Wild-type | Sample source:Total RNA	Genotype:TK0008 deletion | Sample source:Total RNA	Genotype:TK0008 deletion | Sample source:Total RNA
N4-acetylcytidine_1	0.04173007	0.040967448		0.039976959	0.039044422		0.067573162		0.060377669	
N4-acetylcytidine_2	0.038615789	0.037779635		0.03651396	0.036235049		0.057041041		0.053758882	
N4-acetyl-2'-O-methylcytidine_1	0.000457793	0.000399256		0.000399837	0.000397614		0.0002368	0.000241969	0.000151257	0.000264247
N4-acetyl-2'-O-methylcytidine_2	0.000277255	0.000580169		0.000555706	0.000560571		2.56821E-05		7.26955E-05	
N6-acetyladenosine_1	1.64291E-05	4.13197E-05		1.9822E-06	3.92048E-06		6.66736E-06		5.25248E-06	
N6-acetyladenosine_2	5.47636E-06	7.18604E-06		3.96441E-06	9.80119E-06		4.38066E-06		3.54916E-05	
3-(3-amino-3-carboxypropyl)uridine	0.000256674	0.000497636		0.000335233	0.000127755		0.001284482		0.005858175	
2'-O-methyladenosine	0.008134222	0.008756187		0.009052725	0.008638771		0.005168027		0.005935408	
5-carboxycytidine	0.003739716	0.003374964		0.003740851	0.003519864		3.86271E-06		7.30909E-05	
2'-O-methylcytidine	0.043451629	0.042976207		0.044598807	0.04518463		0.039935892		0.038856827	
5-carboxymethyluridine_1	0.000256674	0.000311023		0.000134093	0.000255509		0.000537755		0.002552776	
5-carboxymethyluridine_2	0.000449179	0.000311023		0.000268186	0.000447141		0.001401975		0.00102231	
5-carboxymethylaminomethyluridine_1	0.00038501	0.000746454		0.000134093	0.000319387		0.032550488		0.027296486	
5-carboxymethylaminomethyluridine_2	0.000513347	0.000186614		0.000134093	0		0.00076061		0.000341394	
5-carboxymethylaminomethyluridine_3	0.000192505	0.000248818		0.000536373	0.000255509		0.011215941		0.021612058	
dihydrouridine	0.000962526	0.000124409		0.000536373	0.001085915		0.009189459		0.017594828	
5-formylcytidine	5.15823E-05	1.87151E-05		6.7769E-06	1.95548E-05		7.55739E-06		4.17998E-05	
N6-formyladenosine_1	1.46036E-05	7.18604E-06		3.96441E-06	3.92048E-06		7.05925E-06		2.89397E-06	
N6-formyladenosine_2	3.65091E-06	1.43721E-05		8.32526E-05	7.84095E-06		0.000200453		0.000422307	
2'-O-methylguanosine	0.021204326	0.020632869		0.02190187	0.02108349		0.031536829		0.031779101	
5-hydroxymethylcytidine	2.57911E-05	6.23838E-06		3.38845E-05	3.25913E-05		3.71124E-05		5.77852E-05	
N6-hydroxymethyladenosine_1	9.12727E-06	4.13197E-05		1.38754E-05	3.13638E-05		6.06923E-05		3.72257E-05	
N6-hydroxymethyladenosine_2	5.47636E-06	1.25756E-05		1.38754E-05	5.88072E-06		3.37025E-05		2.00787E-05	
5-hydroxyuridine	0.000834189	0.000248818		0.00020114	0.000191632		0.0001455		0.000985824	
N6-isopentenyladenosine_1	0.002928028	0.003194194		0.00311206	0.003112859		0.003739477		0.0051278	
N6-isopentenyladenosine_2	0.003309548	0.003203176		0.00356202	0.003655845		0.003582198		0.006914863	
2'-O-methylinosine_1	1.27782E-05	3.95232E-05		4.55907E-05	3.72445E-05		9.18268E-06		3.12829E-06	
2'-O-methylinosine_2	1.82545E-05			5.35195E-05	1.56819E-05		8.80511E-06		7.83998E-06	
4-demethylwyosine	9.10643E-05	0.000100414	0.000138869	0.000295306	0.000229831	0.000406414	1.44936E-05		4.42693E-06	
isowyosine	3.64257E-05	0.000113511	6.44116E-05	8.85918E-05	0.000126642	0.000192566	5.00764E-05		5.37592E-05	
N6-(cis-hydroxyisopentenyl)adenosine	1.27782E-05	5.38953E-06		9.91102E-06	7.84095E-06		4.16481E-06		6.86069E-06	
1-methyladenosine	0.067883149	0.069149443		0.070982717	0.073259994		0.009721654		0.010240487	
1,2'-O-dimethyladenosine	1.27782E-05	3.59302E-06		1.78398E-05	1.96024E-05		1.11631E-06		8.81893E-06	
1-methylguanosine	0.004685259	0.004566649		0.003474769	0.002978424		0.009685758		0.009206266	
1-methylinosine	0.002787468	0.002818723		0.002949519	0.002987404		0.002257439		0.002275394	
1-methylpseudouridine_1	0.003914271	0.0013685	0.005653241	0.004827355	0.004024273	0.005774657	0.00201983	0.004927128	0.001018367	0.005353923
1-methylpseudouridine_2	0.002117556	0.001181886		0.002078445	0.004152028		0.00047786		0.001081337	
N2,N2,7-trimethylguanosine	0.001789414	0.001296649		0.002283701	0.001791745		0.002589223		0.004092357	
N2,N2-dimethylguanosine	0.007203187	0.006858705		0.007190704	0.00674015		0.017433942		0.019143562	
N2,7-dimethylguanosine	0.023831531	0.024269598		0.025494761	0.024854597		0.007354044		0.006834521	
2,8-dimethyladenosine	1.82545E-06	1.07791E-05		5.94661E-06	5.88072E-06		7.4652E-07		5.08345E-06	
2-methyladenosine	4.92873E-05	6.64708E-05		5.35195E-05	0.000103893		9.92653E-06		1.99083E-05	
N2-methylguanosine	0.003419465	0.002916368		0.003272976	0.003273921		0.004416371		0.004283025	
N2,2'-O-dimethylguanosine	0.000546386	0.000611215		0.000684126	0.000680113		0.000869057		0.000912969	
3-methylcytidine	0.000148299	3.74303E-05		4.74383E-05	2.60731E-05		1.81081E-05		2.01278E-05	
3-methylpseudouridine_1	0.001475873	0.003607863	0.003384274	0.000938652	0.004535292	0.003538704	0.002780665	0.007573646	0.000935833	0.007207503
3-methylpseudouridine_2	0.000192505	0.00068425		0.004022796	0.000255509		0.003037153		0.001875962	
3-methyluridine	0.003465092	0.003359045		0.003687563	0.00312999		0.007910783	0.009787266	0.003939256	0.010256268
3,2'-O-dimethyluridine	0.000449179	6.22045E-05		0.001206839	0.000319387		0.003508475		0.000343269	
N4,N4-dimethylcytidine	0.004358703	0.004485396		0.00426945	0.004243392		0.005027255		0.004859784	
N4,N4,2'-O-trimethylcytidine	2.57911E-05	3.11919E-05		3.38845E-05	6.51827E-06		0.00032372		0.000348229	
N4-methylcytidine_1	0.025320455	0.024248587		0.025271076	0.025043184		0.02909464		0.027192949	
N4-methylcytidine_2	6.44779E-06	9.98141E-05		8.13229E-05	2.60731E-05		2.40617E-05		2.18516E-05	
N4,2'-O-dimethylcytidine	1.93434E-05	6.23838E-06		1.35538E-05	6.51827E-06		1.7782E-05		1.23394E-05	
5-methylcytidine	0.024424213	0.023868046		0.024349417	0.024560832		0.028128266		0.026587521	
5,2'-O-dimethylcytidine	7.73734E-05	3.74303E-05		5.42152E-05	3.91096E-05		4.46266E-05		3.56687E-05	
5-methyldihydrouridine	0.000192505	0.000870863		0.001609118	0.001022038		7.66907E-05		0.001190302	
5-methyl-2-thiouridine	0.012127823	0.012440906		0.012269527	0.013478122		0.013671064	0.013575267	0.013607687	0.015116634
5-methyluridine	0.003015914	0.002861408	0.003846151	0.00295005	0.0033855	0.003021036	0.009815802	0.003011442	0.005411564	0.00331162
5,2'-O-dimethyluridine	0.000449179	0.000124409		0.000268186	6.38774E-05		0.000448347		0.001291923	
N6,N6-dimethyladenosine	0.024565131	0.023505529		0.025903439	0.02531452		0.006794902		0.007696813	
N6,N6,2'-O-trimethyladenosine	0	5.38953E-06		1.9822E-06	3.92048E-06		2.5405E-06		2.69682E-06	
N6-methyladenosine	0.001538858	0.001584521		0.000761166	0.000678243		0.000784549		0.000704193	
N6,2'-O-dimethyladenosine	0.000567716	0.00049943		0.000555017	0.000523384		0.00083617		0.001119812	
O6-methylguanine	3.18725E-05	6.54873E-05		8.85918E-05	3.75235E-05		5.00677E-05		0.000438939	
N6-methyl-N6-threonylcarbamoyladenosine_1	2.008E-05	5.38953E-06		7.92882E-06	7.84095E-06		7.10053E-06		1.94424E-06	
N6-methyl-N6-threonylcarbamoyladenosine_2	1.82545E-06	1.79651E-06		0	1.96024E-06		7.78453E-07		2.41478E-06	
7-methylguanosine	0.009698349	0.009045981		0.000438037	0.000464353		0.00249959		0.000109108	
8-methyladenosine	0.001478618	0.001541405		0.000699718	0.000588072		0.000798338		0.000795856	
5-methoxycarbonylmethyl-2-thiouridine_1	0.004620123	0.000933068		0.000938652	0.005110188		0.012951856		0.007490915	
5-methoxycarbonylmethyl-2-thiouridine_2	0.004427618	0.001617318		0.002614817	0.002682849		0.000661092		0.002710495	
5-methoxycarbonylmethyluridine_1	0.000128337	0.000186614		0.000469326	0.000383264		0.00020678		0.001278691	
5-methoxycarbonylmethyluridine_2	0.000449179	0.000435432		0.00040228	0.001852443		0.001333586		0.001097578	
5-methoxycarbonylmethyl-2'-O-methyluridine	0.000256674	6.22045E-05		0.00020114	0.000255509		0.001132372		0.002831358	
uridine 5-oxyacetic acid methyl ester_1	0.001347536	0.000559841		0.000603419	0.000255509		0.001660617		0.001714568	
uridine 5-oxyacetic acid methyl ester_2	0.002566735	0.002239363		0.002279584	0.000319387		0.002297748		0.000663758	
uridine 5-oxyacetic acid methyl ester_3	0.001283368	0.001617318		0.000871606	0.000447141		0.001635388		0.001944828	
5-methylaminomethyl-2-thiouridine_1	0.00038501	0.000248818		0.000335233	0.000255509		0.000814612		0.004911714	
5-methylaminomethyl-2-thiouridine_2	0.00038501	0.000559841		0.00020114	0.000702651		0.00145339		0.001246108	
5-methoxyuridine	0.000320842	0.000124409		0.000536373	0.000319387		0.001118978		0.001346451	
2-methylthio-N6-isopentenyladenosine_1	8.57963E-05	9.8808E-05		0.000118932	3.92048E-05		4.52324E-05		4.61705E-05	
2-methylthio-N6-isopentenyladenosine_2	3.28582E-05	1.79651E-05		3.76619E-05	4.70457E-05		0.000350928		0.000669615	
2-methylthio-N6-methyladenosine	2.19054E-05	3.59302E-06		3.17153E-05	7.84095E-06		7.90676E-07		1.51904E-06	
5-carbamoylmethyluridine_1	0.000192505	0.000248818		6.70466E-05	0.000127755		0.00049087		0.000634661	
5-carbamoylmethyluridine_2	6.41684E-05	0.000622045		0.00040228	0.000255509		0.00062622		0.000564849	
pseudouridine	0.043313655	0.045533715		0.046195106	0.044267007		0.055201024		0.054539234	
adenosine	1	1		1	1		1		1	
cytidine	1	1		1	1		1		1	
guanosine	1	1		1	1		1		1	
inosine	6.024E-05	8.62325E-05	0.000141425	4.55907E-05	0.000109773	7.33927E-05	2.96535E-06	3.42698E-06	1.15398E-05	3.65878E-06
uridine	1	1		1	1		1		1	
2-thiocytidine	0.003043355	0.00258269		0.003151261	0.003154841		0.005909035		0.005401687	
2-thiouridine	0.00038501	0.000311023		0.000335233	6.38774E-05		0.000553075		0.001202801	
2-thio-2'-O-methyluridine	0.000513347	0.000746454		0.000469326	0.000255509				0.001298097	
4-thiouridine	0.00237423	0.002177158		0.004022796	0.003577132		0.004728912	0.004880689	0.004520402	0.005719155
N6-threonylcarbamoyladenosine_1	0.000124131	6.82674E-05		8.91992E-05	6.27276E-05		0.000113137		0.000214434	
N6-threonylcarbamoyladenosine_2	0.000186196	0.000167075		0.000138754	6.86083E-05		0.000197744		0.000266451	
2'-O-methyluridine	0.030672485	0.03172431		0.031243714	0.031236027		0.02682181		0.027472608	
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Mass_Transition
N4-acetylcytidine_1	286.1 -> 154.1
N4-acetylcytidine_2	286.1 -> 112.1
N4-acetyl-2'-O-methylcytidine_1	300.1 -> 154.1
N4-acetyl-2'-O-methylcytidine_2	322.1 -> 176.1
N6-acetyladenosine_1	310.1 -> 178.2
N6-acetyladenosine_2	332.1 -> 200.2
3-(3-amino-3-carboxypropyl)uridine	346.1 -> 214.1
2'-O-methyladenosine	282.1 -> 136.1
5-carboxycytidine	288.1 -> 156.1
2'-O-methylcytidine	258.1 -> 112.1
5-carboxymethyluridine_1	303.1 -> 171.1
5-carboxymethyluridine_2	303.1 -> 125.1
5-carboxymethylaminomethyluridine_1	332.1 -> 200.1
5-carboxymethylaminomethyluridine_2	332.1 -> 125.1
5-carboxymethylaminomethyluridine_3	354.1 -> 222.1
dihydrouridine	247.1 -> 115.0
5-formylcytidine	272.1 -> 140.1
N6-formyladenosine_1	296.1 -> 164.1
N6-formyladenosine_2	296.1 -> 136.1
2'-O-methylguanosine	298.1 -> 152.1
5-hydroxymethylcytidine	274.1 -> 142.1
N6-hydroxymethyladenosine_1	298.1 -> 166.1
N6-hydroxymethyladenosine_2	298.1 -> 136.1
5-hydroxyuridine	261.1 -> 129.1
N6-isopentenyladenosine_1	336.1 -> 136.1
N6-isopentenyladenosine_2	336.1 -> 204.2
2'-O-methylinosine_1	283.1 -> 137.1
2'-O-methylinosine_2	305.1 -> 159.1
4-demethylwyosine	344.1 -> 212.1
isowyosine	336.1 -> 204.2
N6-(cis-hydroxyisopentenyl)adenosine	352.1 -> 220.2
1-methyladenosine	282.1 -> 150.1
1,2'-O-dimethyladenosine	296.1 -> 150.1
1-methylguanosine	298.1 -> 166.2
1-methylinosine	283.1 -> 151.1
1-methylpseudouridine_1	259.1 -> 223.1
1-methylpseudouridine_2	259.1 -> 139.1
N2,N2,7-trimethylguanosine	326.1 -> 194.1
N2,N2-dimethylguanosine	312.1 -> 180.2
N2,7-dimethylguanosine	312.1 -> 180.1
2,8-dimethyladenosine	296.1 -> 164.1
2-methyladenosine	282.1 -> 150.2
N2-methylguanosine	298.1 -> 166.2
N2,2'-O-dimethylguanosine	312.1 -> 166.1
3-methylcytidine	258.1 -> 126.1
3-methylpseudouridine_1	259.1 -> 223.1
3-methylpseudouridine_2	259.1 -> 139.1
3-methyluridine	259.1 -> 127.1
3,2'-O-dimethyluridine	273.1 -> 127.1
N4,N4-dimethylcytidine	272.1 -> 140.2
N4,N4,2'-O-trimethylcytidine	286.1 -> 140.2
N4-methylcytidine_1	258.1 -> 126.1
N4-methylcytidine_2	258.1 -> 95.0
N4,2'-O-dimethylcytidine	272.1 -> 126.1
5-methylcytidine	258.1 -> 126.1
5,2'-O-dimethylcytidine	272.1 -> 126.1
5-methyldihydrouridine	261.1 -> 129.1
5-methyl-2-thiouridine	275.1 -> 143.1
5-methyluridine	259.1 -> 127.1
5,2'-O-dimethyluridine	273.1 -> 127.1
N6,N6-dimethyladenosine	296.1 -> 164.2
N6,N6,2'-O-trimethyladenosine	310.1 -> 164.2
N6-methyladenosine	282.1 -> 150.1
N6,2'-O-dimethyladenosine	296.1 -> 150.1
O6-methylguanine	298.1 -> 166.2
N6-methyl-N6-threonylcarbamoyladenosine_1	427.1 -> 295.2
N6-methyl-N6-threonylcarbamoyladenosine_2	427.1 -> 150.1
7-methylguanosine	298.1 -> 166.2
8-methyladenosine	282.1 -> 150.1
5-methoxycarbonylmethyl-2-thiouridine_1	333.1 -> 201.1
5-methoxycarbonylmethyl-2-thiouridine_2	355.1 -> 223.1
5-methoxycarbonylmethyluridine_1	317.1 -> 185.2
5-methoxycarbonylmethyluridine_2	339.1 -> 207.2
5-methoxycarbonylmethyl-2'-O-methyluridine	331.1 -> 185.1
uridine 5-oxyacetic acid methyl ester_1	333.1 -> 201.1
uridine 5-oxyacetic acid methyl ester_2	333.1 -> 141.1
uridine 5-oxyacetic acid methyl ester_3	355.1 -> 223.1
5-methylaminomethyl-2-thiouridine_1	304.1 -> 172.1
5-methylaminomethyl-2-thiouridine_2	304.1 -> 141.0
5-methoxyuridine	275.1 -> 143.1
2-methylthio-N6-isopentenyladenosine_1	382.1 -> 250.2
2-methylthio-N6-isopentenyladenosine_2	382.1 -> 182.1
2-methylthio-N6-methyladenosine	328.1 -> 196.1
5-carbamoylmethyluridine_1	302.1 -> 170.1
5-carbamoylmethyluridine_2	302.1 -> 125.1
pseudouridine	245.1 -> 209.1
adenosine	268.1 -> 136.1
cytidine	244.1 -> 112.1
guanosine	284.1 -> 152.1
inosine	291.1 -> 159.1
uridine	245.1 -> 113.0
2-thiocytidine	260.1 -> 128.1
2-thiouridine	261.1 -> 129.1
2-thio-2'-O-methyluridine	275.1 -> 129.0
4-thiouridine	261.1 -> 129.1
N6-threonylcarbamoyladenosine_1	413.1 -> 136.1
N6-threonylcarbamoyladenosine_2	413.1 -> 281.2
2'-O-methyluridine	259.1 -> 113.0
METABOLITES_END
#END