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A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs (2015)

Taoka, M., Nobe, Y., Hori, M., Takeuchi, A., Masaki, S., Yamauchi, Y., Nakayama, H., Takahashi, N., Isobe, T.

Oxford University Press

In: Nucleic Acids Research

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Publication Date: 2015-10-15

Description: We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA.

Keywords: Nucleic acid modification, RNA characterisation and manipulation

Print ISSN: 0305-1048

Electronic ISSN: 1362-4962

Topics: Biology

Published by Oxford University Press

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A mass spectrometry-based method for direct determination of pseudouridine in RNA (2016)

Yamauchi, Y., Nobe, Y., Izumikawa, K., Higo, D., Yamagishi, Y., Takahashi, N., Nakayama, H., Isobe, T., Taoka, M.

Oxford University Press

In: Nucleic Acids Research

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Publication Date: 2016-04-08

Description: Pseudouridine (5-ribosyluracil, ) is the only ‘mass-silent’ nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of in RNA. The method assigns a -containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C 9 H 7 N 2 O 4 ] 1– , m/z 207.04) produced by collision-induced dissociation MS, and it determines the -containing nucleotide sequence by pseudo-MS 3 , i.e. in-source fragmentation followed by MS 2 . By applying this method, we identified all of the known s in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown s in the U5 and U6 snRNAs. Because the method allows direct determination of in a subpicomole quantity of RNA, it will serve as a useful tool for the structure/function studies of a wide variety of non-coding RNAs.

Keywords: Nucleic acid modification, RNA characterisation and manipulation

Print ISSN: 0305-1048

Electronic ISSN: 1362-4962

Topics: Biology

Published by Oxford University Press

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Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase (2016)

Aschenbrenner, J., Marx, A.

Oxford University Press

In: Nucleic Acids Research

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Publication Date: 2016-05-06

Description: Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.

Keywords: Nucleic acid modification, RNA characterisation and manipulation

Print ISSN: 0305-1048

Electronic ISSN: 1362-4962

Topics: Biology

Published by Oxford University Press

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Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity (2016)

Krogh, N., Jansson, M. D., Häfner, S. J., Tehler, D., Birkedal, U., Christensen-Dalsgaard, M., Lund, A. H., Nielsen, H.

Oxford University Press

In: Nucleic Acids Research

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Publication Date: 2016-09-20

Description: Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2'- O -Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2'- O -Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2'- O -Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.

Keywords: Nucleic acid modification, RNA characterisation and manipulation

Print ISSN: 0305-1048

Electronic ISSN: 1362-4962

Topics: Biology

Published by Oxford University Press

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Complementary-addressed site-directed spin labeling of long natural RNAs (2016)

Babaylova, E. S., Malygin, A. A., Lomzov, A. A., Pyshnyi, D. V., Yulikov, M., Jeschke, G., Krumkacheva, O. A., Fedin, M. V., Karpova, G. G., Bagryanskaya, E. G.

Oxford University Press

In: Nucleic Acids Research

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Publication Date: 2016-09-20

Description: Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of 330 nucleotides and having a complicated spatial structure. Application of pulsed double electron–electron resonance provided spin–spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.

Keywords: Nucleic acid modification, RNA characterisation and manipulation

Print ISSN: 0305-1048

Electronic ISSN: 1362-4962

Topics: Biology

Published by Oxford University Press

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