Summary of Study ST003725

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002314. The data can be accessed directly via it's Project DOI: 10.21228/M8HR7S This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003725
Study TitleIdentification of temperature-sensitive modifications in Thermococcus kodakarensis
Study SummaryNucleoside analysis of total RNA and RNA subfractions from T. kodakarensis grown at 65, 75, 85 and 95 degree Celcius.
Institute
New England Biolabs
Last NameTsai
First NameYueh-Lin
Address44 Dunham Ridge, Beverly, MA 01915
Emailatsai@neb.com
Phone978-380-6587
Submit Date2025-02-01
Raw Data AvailableYes
Raw Data File Type(s)mzML, d
Analysis Type DetailLC-MS
Release Date2025-02-14
Release Version1
Yueh-Lin Tsai Yueh-Lin Tsai
https://dx.doi.org/10.21228/M8HR7S
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002314
Project DOI:doi: 10.21228/M8HR7S
Project Title:Comprehensive Nucleoside Analysis of Archaeal RNA Modification Profiles Reveals a m7G in the Conserved P-loop of 23S rRNA
Project Summary:Extremophilic archaea employ diverse chemical RNA modifications, providing a rich source of new enzymes for biotechnologically valuable RNA manipulations. Our understanding of the modified nucleoside profiles in Archaea, as well as the functions and dynamic regulation of specific RNA modifications is far from complete. Here, we established an extensive profile of nucleoside modifications in thermophilic and mesophilic Archaea through highly sensitive LC-MS/MS analysis and rigorous non-coding RNA depletion, identifying - with high confidence - at least four previously unannotated modifications in archaeal mRNAs. Nucleoside quantification analysis conducted on total, large, small, and mRNA-enriched subfractions of the model hyperthermophilic archaeon Thermococcus kodakarensis revealed a series of modifications whose abundance is dynamically responsive to growth temperatures, implying that specific RNA modifications are fitness relevant under specific growth conditions. To predict the RNA-modifying enzymes most likely to generate the new and dynamic RNA modifications, we leveraged a bioinformatics analysis of open-access databases to annotate likely functional domains of archaeal proteins. Putative enzyme activities were confirmed in vitro and in vivo by assessing the presence of the target RNA modification in genetic deletion strains of T. kodakarensis. Our approach led to the discovery of a methyltransferase-encoded gene responsible for m7G modification in the P-loop of 23S rRNA peptidyl transferase center and validates a novel and effective platform for discovering RNA-modifying enzymes through LC-MS/MS analysis that will accelerate efforts of the community towards uncovering the complex and dynamic roles of RNA modifications.
Institute:New England Biolabs
Last Name:Tsai
First Name:Yueh-Lin
Address:44 Dunham Ridge, Beverly, MA 01915
Email:atsai@neb.com
Phone:978-380-6587

Subject:

Subject ID:SU003857
Subject Type:Archaea
Subject Species:Thermococcus kodakarensis
Taxonomy ID:311400

Factors:

Subject type: Archaea; Subject species: Thermococcus kodakarensis (Factor headings shown in green)

mb_sample_id local_sample_id Growth temperature
SA406949Tk_large_AU_65C_rep2-r00165C
SA406950Tk_large_AU_65C_rep3-r00265C
SA406951Tk_large_AU_65C_rep3-r00165C
SA406952Tk_large_pseudoU_65C_rep2-r00165C
SA406953Tk_total_AU_65C_rep1-r00265C
SA406954Tk_large_GC_65C_rep2-r00265C
SA406955Tk_large_GC_65C_rep2-r00165C
SA406956Tk_large_AU_65C_rep2-r00265C
SA406957Tk_large_pseudoU_65C_rep1-r00165C
SA406958Tk_large_GC_65C_rep3-r00265C
SA406959Tk_large_GC_65C_rep1-r00265C
SA406960Tk_large_GC_65C_rep1-r00165C
SA406961Tk_large_AU_65C_rep1-r00265C
SA406962Tk_large_AU_65C_rep1-r00165C
SA406963Tk_total_pseudoU_65C_rep3-r00165C
SA406964Tk_total_GC_65C_rep3-r00265C
SA406965Tk_total_GC_65C_rep3-r00165C
SA406966Tk_large_GC_65C_rep3-r00165C
SA406967Tk_large_pseudoU_65C_rep3-r00165C
SA406968Tk_total_AU_65C_rep3-r00165C
SA406969Tk_small_GC_65C_rep2-r00265C
SA406970Tk_mRNA_85C_rep165C
SA406971Tk_mRNA_75C_rep165C
SA406972Tk_mRNA_65C_rep165C
SA406973Tk_small_pseudoU_65C_rep3-r00165C
SA406974Tk_small_pseudoU_65C_rep2-r00165C
SA406975Tk_small_GC_65C_rep3-r00265C
SA406976Tk_small_GC_65C_rep3-r00165C
SA406977Tk_small_GC_65C_rep2-r00165C
SA406978Tk_small_AU_65C_rep1-r00165C
SA406979Tk_small_AU_65C_rep3-r00265C
SA406980Tk_small_AU_65C_rep3-r00165C
SA406981Tk_small_AU_65C_rep2-r00265C
SA406982Tk_small_AU_65C_rep2-r00165C
SA406983Tk_small_pseudoU_65C_rep1-r00165C
SA406984Tk_small_GC_65C_rep1-r00265C
SA406985Tk_small_GC_65C_rep1-r00165C
SA406986Tk_small_AU_65C_rep1-r00265C
SA406987Tk_total_AU_65C_rep3-r00265C
SA406988Tk_total_AU_65C_rep1-r00165C
SA406989Tk_total_GC_65C_rep1-r00265C
SA406990Tk_total_GC_65C_rep2-r00265C
SA406991Tk_total_GC_65C_rep1-r00165C
SA406992Tk_total_pseudoU_65C_rep1-r00165C
SA406993Tk_total_pseudoU_65C_rep1-r00265C
SA406994Tk_total_AU_65C_rep2-r00165C
SA406995Tk_total_GC_65C_rep2-r00165C
SA406996Tk_total_AU_65C_rep2-r00265C
SA406997Tk_total_pseudoU_65C_rep2-r00165C
SA406998Tk_total_pseudoU_75C_rep2-r00175C
SA406999Tk_small_GC_75C_rep3-r00275C
SA407000Tk_large_AU_75C_rep2-r00275C
SA407001Tk_total_AU_75C_rep2-r00275C
SA407002Tk_large_GC_75C_rep2-r00175C
SA407003Tk_large_GC_75C_rep2-r00275C
SA407004Tk_total_AU_75C_rep2-r00175C
SA407005Tk_total_GC_75C_rep1-r00175C
SA407006Tk_large_pseudoU_75C_rep2-r00175C
SA407007Tk_small_pseudoU_75C_rep2-r00175C
SA407008Tk_large_AU_75C_rep3-r00175C
SA407009Tk_large_AU_75C_rep3-r00275C
SA407010Tk_large_GC_75C_rep3-r00175C
SA407011Tk_large_GC_75C_rep3-r00275C
SA407012Tk_large_pseudoU_75C_rep3-r00175C
SA407013Tk_total_pseudoU_75C_rep1-r00175C
SA407014Tk_total_pseudoU_75C_rep1-r00275C
SA407015Tk_small_pseudoU_75C_rep3-r00175C
SA407016Tk_small_AU_75C_rep1-r00175C
SA407017Tk_small_AU_75C_rep1-r00275C
SA407018Tk_small_GC_75C_rep3-r00175C
SA407019Tk_small_GC_75C_rep1-r00175C
SA407020Tk_small_GC_75C_rep1-r00275C
SA407021Tk_small_pseudoU_75C_rep1-r00175C
SA407022Tk_small_AU_75C_rep2-r00175C
SA407023Tk_small_AU_75C_rep2-r00275C
SA407024Tk_small_GC_75C_rep2-r00275C
SA407025Tk_small_GC_75C_rep2-r00175C
SA407026Tk_total_GC_75C_rep1-r00275C
SA407027Tk_small_AU_75C_rep3-r00175C
SA407028Tk_small_AU_75C_rep3-r00275C
SA407029Tk_large_AU_75C_rep2-r00175C
SA407030Tk_total_AU_75C_rep3-r00175C
SA407031Tk_large_AU_75C_rep1-r00175C
SA407032Tk_total_GC_75C_rep3-r00275C
SA407033Tk_total_AU_75C_rep3-r00275C
SA407034Tk_large_AU_75C_rep1-r00275C
SA407035Tk_large_GC_75C_rep1-r00175C
SA407036Tk_large_GC_75C_rep1-r00275C
SA407037Tk_mRNA_75C_rep275C
SA407038Tk_total_pseudoU_75C_rep3-r00175C
SA407039Tk_total_AU_75C_rep1-r00175C
SA407040Tk_total_AU_75C_rep1-r00275C
SA407041Tk_total_GC_75C_rep3-r00175C
SA407042Tk_mRNA_95C_rep175C
SA407043Tk_mRNA_65C_rep275C
SA407044Tk_total_GC_75C_rep2-r00275C
SA407045Tk_large_pseudoU_75C_rep1-r00175C
SA407046Tk_total_GC_75C_rep2-r00175C
SA407047Tk_total_pseudoU_85C_rep3-r00185C
SA407048Tk_small_AU_85C_rep1-r00285C
Showing page 1 of 2     Results:    1  2  Next     Showing results 1 to 100 of 196

Collection:

Collection ID:CO003850
Collection Summary:T. kodakarensis was grown at 65C, 75C, 85C, or 95C in anaerobic artificial sea water supplemented with yeast extract and tryptone to mid-exponential phase (Optical density ~0.3) in triplicates before harvest. Harvested archaeal cell pellets were resuspended in 10 mL of TRI reagent (Molecular Research Center, Inc., Cat #TR118). The resuspended cells were homogenized using a beads beater at 4.0 m/s for 20 seconds x 2 cycles (MP Biomedicals, FastPrep-24TM). Subsequently, the mixture was centrifuged at 14000 g for 5 minutes to precipitate any cell debris. Supernatants were collected post-centrifugation and treated with 50 μL of BAN reagent (Molecular Research Center, Inc., Cat #BN191) per mL of supernatant for aqueous-organic phase separation. RNA from the aqueous phase was isolated by isopropanol precipitation and subjected to DNase I treatment (NEB, Cat #M0303S) to remove genomic DNA contamination. To further purify the DNase I-treated RNA, an equal volume of acid phenol-chloroform with isoamyl alcohol (125:24:1, Thermo Fisher Scientific, Cat #AM9722) was added to the reaction and centrifuged at 21300 g for 2 minutes to separate the aqueous phase from the organic phase. The aqueous phase containing RNA was then precipitated with 1.5 volumes of isopropanol and 0.1 volume of sodium acetate (Sigma Aldrich, Cat #S7899) at –20°C overnight. Finally, the precipitated RNA pellets were washed with 75% ethanol and dissolved in nuclease-free water.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR003866
Treatment Summary:T. kodakarensis were grown at 65, 75, 85, or 95C in anaerobic artificial sea water to OD ~0.3.

Sample Preparation:

Sampleprep ID:SP003863
Sampleprep Summary:We separated total RNA recovered from T. kodakarensis cultures grown at each temperature into large (> 200 nt), small (< 200 nt) using the RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017). mRNA-enriched fractions were prepared using the NEBNext rRNA Depletion Kit (NEB, Cat #E7850X) with the following changes: The NEBNext rRNA depletion solutions provided in the kit were substituted for customized DNA probe mixtures (at 1 uM for each probe) fully complementary to rRNA sequences corresponding to T. kodakarensis.

Chromatography:

Chromatography ID:CH004640
Chromatography Summary:Solvent A is pH 4.5
Instrument Name:Agilent 1290 Infinity II UHPLC
Column Name:Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um)
Column Temperature:30
Flow Gradient:1%-40% Solvent B in 26.5 min
Flow Rate:0.6 mL/min
Solvent A:100% water; 10mM ammonium acetate
Solvent B:100% methanol
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006111
Analysis Type:MS
Chromatography ID:CH004640
Num Factors:4
Num Metabolites:98
Rt Units:Minutes
Units:Integrative area under chromatographic peak
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