Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells

Basanta‐Sanchez, Maria; Temple, Sally; Ansari, Suraiya A.; D'Amico, A.; Agris, Paul F.

doi:10.1093/nar/gkv971

Cited by 112 publications

(118 citation statements)

References 49 publications

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“…Although quadruple time of flight (Q-TOF) and Orbitrap high mass accuracy mass spectrometers are sufficient for relative quantification, the use of a tandem QQQ LC-MS/MS with ESI is advised for absolute nucleoside quantification, as its capacity for sensitivity is superior to that of other instrumentation. Supplementation of an additional and complementary Q-TOF MS/MS method and/or various nuclear magnetic resonance (NMR) spectroscopy techniques can also aid in structural assignment [116,152,153]. …”

Section: Methods For Investigation and Quantification Of Trna Modimentioning

confidence: 99%

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

2017

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Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes.

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Section: Methods For Investigation and Quantification Of Trna Modimentioning

confidence: 99%

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

2017

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“…Briefly, digestion was performed with nuclease P1 (Sigma, 2U) in buffer containing 25 mM NaCl and 2.5 mM ZnCl 2 for 2 h at 37°C, followed by incubation with Antarctic Phosphatase (NEB, 5U) for an additional 2 h at 37°C. Nucleosides were then separated and quantified at the Duke Molecular Physiology Institute using UPLC-MS/MS as previously described (Basanta-Sanchez et al 2016), except acetic acid replaced formic acid in the mobile phase.…”

Section: Lc-ms/ms Of Poly(a)mentioning

confidence: 99%

N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Fry

Law

Ilkayeva

et al. 2017

RNA

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Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by N 6 -methyladenosine (m 6 A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m 6 A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m 6 A. Our results show hypoxia increases m 6 A content of poly(A) + messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m 6 A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m 6 A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m 6 A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m 6 A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m 6 A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress.

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“…In an effort to verify this, we replicated the neural differentiation protocol as previously used by us and others [11, 12, 15, 16]. Briefly, the H9 cells were grown on feeder-free conditions on Matrigel-coated tissue culture dishes in mTeSR1 media until the cells were fully confluent.…”

Section: Resultsmentioning

confidence: 99%

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

Varghese

Parween

Ardah

et al. 2017

Stem Cells International

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Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results.

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Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells

Cited by 112 publications

References 49 publications

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

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Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells

Cited by 112 publications

References 49 publications

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs

Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

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N⁶-methyladenosine is required for the hypoxic stabilization of specific mRNAs