Summary of Study ST003727
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 ID | ST003727 |
| Study Title | Identification of modified nucleosides in mRNA-enriched archaeal samples |
| Study Summary | Total RNA extracted from five archaeal species were depleted with rRNAs and digested to nucleosides for UHPLC-QqQ analysis. |
| Institute | New England Biolabs |
| Last Name | Tsai |
| First Name | Yueh-Lin |
| Address | 44 Dunham Ridge, Beverly, MA 01915 |
| atsai@neb.com | |
| Phone | 978-380-6587 |
| Submit Date | 2025-02-05 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-02-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU003859 |
| Subject Type: | Cultured cells |
| Subject Species: | Thermococcus kodakarensis, Thermococcus sp. AM4, Methanococcus maripaludis, Sulfolobus acidocaldarius, Sulfolobus islandicus |
| Taxonomy ID: | 311400, 246969, 39152, 2285, 43080 |
Factors:
Subject type: Cultured cells; Subject species: Thermococcus kodakarensis, Thermococcus sp. AM4, Methanococcus maripaludis, Sulfolobus acidocaldarius, Sulfolobus islandicus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Species | Sample source |
|---|---|---|---|
| SA407190 | M_mari_mRNA_rA_rep1-r001 | Methanococcus maripaludis | rRNA_depletion |
| SA407191 | M_mari_mRNA_GC_rep2 | Methanococcus maripaludis | rRNA_depletion |
| SA407192 | M_mari_mRNA_rU_rep2 | Methanococcus maripaludis | rRNA_depletion |
| SA407193 | M_mari_mRNA_GC_rep1-r002 | Methanococcus maripaludis | rRNA_depletion |
| SA407194 | M_mari_mRNA_GC_rep1-r001 | Methanococcus maripaludis | rRNA_depletion |
| SA407195 | M_mari_mRNA_rU_rep1-r002 | Methanococcus maripaludis | rRNA_depletion |
| SA407196 | M_mari_mRNA_rU_rep1-r001 | Methanococcus maripaludis | rRNA_depletion |
| SA407197 | M_mari_mRNA_rA_rep1-r002 | Methanococcus maripaludis | rRNA_depletion |
| SA407198 | M_mari_mRNA_rA_rep2 | Methanococcus maripaludis | rRNA_depletion |
| SA407199 | S_acid_mRNA_rU_rep1-r001 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407200 | S_acid_mRNA_GC_rep2 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407201 | S_acid_mRNA_GC_rep1-r001 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407202 | S_acid_mRNA_rA_rep1 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407203 | S_acid_mRNA_rA_rep1-r001 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407204 | S_acid_mRNA_GC_rep1-r002 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407205 | S_acid_mRNA_rA_rep2 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407206 | S_acid_mRNA_rU_rep2 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407207 | S_acid_mRNA_rU_rep1-r002 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407208 | S_acid_mRNA_rU_rep1-r003 | Sulfolobus acidocaldarius | rRNA_depletion |
| SA407209 | S_island_mRNA_rA_rep1-r002 | Sulfolobus islandicus | rRNA_depletion |
| SA407210 | S_island_mRNA_rU_rep1-r001 | Sulfolobus islandicus | rRNA_depletion |
| SA407211 | S_island_mRNA_rU_rep1-r002 | Sulfolobus islandicus | rRNA_depletion |
| SA407212 | S_island_mRNA_GC_rep1 | Sulfolobus islandicus | rRNA_depletion |
| SA407213 | S_island_mRNA_rA_rep2 | Sulfolobus islandicus | rRNA_depletion |
| SA407214 | S_island_mRNA_rU_rep2 | Sulfolobus islandicus | rRNA_depletion |
| SA407215 | S_island_mRNA_GC_rep2 | Sulfolobus islandicus | rRNA_depletion |
| SA407216 | S_island_mRNA_rA_rep1-r001 | Sulfolobus islandicus | rRNA_depletion |
| SA407217 | Tk_mRNA_mods_rep1_AT_01-r001 | Thermococcus kodakarensis | rRNA_depletion |
| SA407218 | Tk_mRNA_mods_rep1_AT_01-r003 | Thermococcus kodakarensis | rRNA_depletion |
| SA407219 | Tk_mRNA_mods_rep2_AT_01-r001 | Thermococcus kodakarensis | rRNA_depletion |
| SA407220 | Tk_mRNA_mods_rep1_AT_01-r002 | Thermococcus kodakarensis | rRNA_depletion |
| SA407221 | Tk_mRNA_mods_rep2_AT_01-r002 | Thermococcus kodakarensis | rRNA_depletion |
| SA407222 | Tk_mRNA_mods_rep2_AT_01-r003 | Thermococcus kodakarensis | rRNA_depletion |
| SA407223 | TAM4_mRNA_GC_rep1-r002 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407224 | TAM4_mRNA_rA_rep1-r001 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407225 | TAM4_mRNA_rA_rep1-r002 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407226 | TAM4_mRNA_rU_rep1-r001 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407227 | TAM4_mRNA_rU_rep1-r002 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407228 | TAM4_mRNA_GC_rep2 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407229 | TAM4_mRNA_rU_rep2 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407230 | TAM4_mRNA_rA_rep2 | Thermococcus sp. AM4 | rRNA_depletion |
| SA407231 | TAM4_mRNA_GC_rep1-r001 | Thermococcus sp. AM4 | rRNA_depletion |
| Showing results 1 to 42 of 42 |
Collection:
| Collection ID: | CO003852 |
| Collection Summary: | T. kodakarensis strains were grown at 85C in anaerobic artificial sea water supplemented with yeast extract and tryptone to mid-exponential phase (Optical density ~0.3) before harvest. Sources of Thermococcus sp. AM4, Sulfolobus islandicus M16.4, Sulfolobus acidocaldarius, and Methanococcus maripaludis biomasses were contributed by C.S. Raman (University of Maryland), Rachel Whitaker (University of Illinois at Urbana-Champaign), Sonja-Verena Albers (University of Freiburg), and Barney Whitman (University of Georigia), respectively. 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: | Ribonucleic acid |
Treatment:
| Treatment ID: | TR003868 |
| Treatment Summary: | To remove rRNA and tRNA, total RNA was separated into large (> 200 nt) and small RNA (< 200 nt) fractions using the RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017). Subsequently, 50 ug of the large RNA fraction were subjected to rRNA depletion 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 each archaeal species; All volumes for the probe hybridization, RNase H and DNase I digestion reactions were scaled up by fivefold in 10 parallel reactions. Following the enzymatic treatment, the reactions were cleaned up using RNA Clean and Concentrator Kit (Zymo Research, Cat #R1017), the mRNA-enriched fractions were eluted in 10 uL water and combined. |
Sample Preparation:
| Sampleprep ID: | SP003865 |
| Sampleprep Summary: | mRNA-enriched samples were digested to nucleosides at 37°C overnight using a Nucleoside Digestion Mix (NEB, Cat #M0649S). The digested RNAs were subsequently injected without prior purification on an Agilent 1290 Infinity II UHPLC equipped with a G7117 diode array detector and an Agilent 6495C Triple-Quadrupole Mass Spectrometer operating in positive electrospray ionization (+ESI) mode. |
Combined analysis:
| Analysis ID | AN006113 | AN006114 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
| Column | Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) | Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) |
| MS Type | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole |
| MS instrument name | Agilent 6495 QQQ | Agilent 6495 QQQ |
| Ion Mode | POSITIVE | POSITIVE |
| Units | femtomole | femtomole |
Chromatography:
| Chromatography ID: | CH004642 |
| Chromatography Summary: | Solvent A pH is 4.5 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) |
| Column Temperature: | 30 |
| Flow Gradient: | 1%-23% Solvent B in 7.5 min |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | 100% water; 10mM ammonium acetate |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004643 |
| Chromatography Summary: | Solvent A pH is 4.5 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters XSelect HSS T3 XP (100 × 2.1mm, 2.5um) |
| Column Temperature: | 30 |
| Flow Gradient: | 1%-40% Solvent B in 7.5 min |
| Flow Rate: | 0.6mL/min |
| Solvent A: | 100% water; 10mM ammonium acetate |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005819 |
| Analysis ID: | AN006113 |
| Instrument Name: | Agilent 6495 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric data were acquired using dynamic multiple reaction monitoring (DMRM) mode. Identification of each nucleoside species was based on the associated retention time and mass transition in the extracted chromatogram. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005820 |
| Analysis ID: | AN006114 |
| Instrument Name: | Agilent 6495 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Mass spectrometric data were acquired using dynamic multiple reaction monitoring (DMRM) mode. Identification of each nucleoside species was based on the associated retention time and mass transition in the extracted chromatogram. |
| Ion Mode: | POSITIVE |
