Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs(rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation.In ribosome biogenesis,rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes.However,rRNA modifications in plants have yet to be fully defined.Herein,we proposed a method to purify rRNAs by a successive isolation with different strategies,including poly A-based m RNA depletion and agarose gel electrophoresis-based purification,with which highly pure rRNAs could be obtained.In addition,we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA.LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA,respectively.Notably,among these identified modifications,2 kinds of modifications of N^(2),N^(2)-dimethylguanosine(m^(2,2)G)and N^(6),N^(6)-dimethyladenosine(m^(6,6)A) in 18S rRNA,and 4 kinds of modifications of m^(2,2)G,m^(6,6)A,N7-methylguanosine(m^(7)G) and 3-methyluridin(m^(3)U) in 25S rRNA,were first reported to be present in plants.Moreover,exposure of Arabidopsis thaliana to cadmium(Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants,indicating that rRNA modifications play important roles in response to environmental stress.The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.

Adolescent alcohol exposure alters DNA and RNA modifications in peripheral blood by liquid chromatography-tandem mass spectrometry analysis

Alcohol consumption is a critical risk factor contributing to a verity of human diseases. The incidence of alcohol use disorder increases across adolescence in recent years. Accumulating line of evidence suggests that alcohol-induced changes of DNA cytosine methylation(5-methyl-2-deoxycytidine, 5 m C) in genomes play an important role in the development of diseases. However, systemic investigation of the effects of adolescent alcohol exposure on DNA and RNA modifications is still lacked. Especially, there hasn’t been any report to study the effects of alcohol exposure on RNA modifications. Similar to DNA modifications,RNA modifications recently have been identified to function as new regulators in modulating numbers of biological processes. In the current study, we systematically investigated the effects of alcohol exposure on both DNA and RNA modifications in peripheral blood of adolescent rats by liquid chromatographyelectrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) analysis. The developed LC-ESI-MS/MS method enabled the sensitive and accurate determination of 2 DNA modifications and 12 RNA modifications. As for the alcohol exposure experiments, the adolescent rats were intraperitoneally injected with ethanol with an interval of one day for a total 14 days. The quantification results by LC-ESI-MS/MS analysis showed that adolescent alcohol exposure could alter both DNA and RNA modifications in peripheral blood. Specifically, we observed an overall decreased trend of RNA modifications. The discovery of the significant alteration of the levels of DNA and RNA modifications under alcohol exposure indicates that alcohol consumption may increase the risk of the incidence and development of diseases through dysregulating DNA and RNA modifications.

Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR

5-Methylcytosine(5mC)is the most important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC).It has been known that 5mC,5hmC and 5fC play critical roles in modulating gene expression.However,unlike the 5mC,5hmC,and 5fC,the functions of 5caC are still underexplored.Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA.In the current study,we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR(qPCR)for the site-specific quantification of 5caC in DNA.This method depends on the selective conversion of 5caC to form dihydrouracil(DHU)by pyridine borane treatment.DHU behaves like thymine and pairs with adenine(DHU-A).Thus,the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A,which is utilized to achieve the site-specific detection and quantification of 5caC in DNA.As a proof-of-concept,the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples.The method is rapid,straightforward and cost-effective,and shows promising in promoting the investigation of the functional roles of 5caC in future study.

Comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry

RNA molecules contain diverse modifications that display important functions in a variety of physiological and pathological processes.So far over 150 chemical modifications have been characterized to be present in various RNA species,such as in messenger RNA(mRNA),ribosomal RNA(rRNA),and transfer RNA(tRNA).Previous studies revealed that certain RNA modifications were correlated to specific human diseases,indicating RNA modifications could serve as the potential indicator of human diseases.However,systemic investigation of the alteration of RNA modifications in different RNA species of carcinoma tissues are still lacked.Herein,we carried out the comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry(LC-ESIMS/MS)analysis.The developed method allowed us to simultaneously detect 48 different types of RNA modifications.Using this method,we detected 10,15,14,and 25 modifications in m RNA,18 S r RNA,28 S rRNA and small RNA(<200 nt),respectively.Compared to the normal tissues,we revealed a total of 14 RNA modification exhibited significant increase and 2 RNA modifications showed significant decrease in thyroid carcinoma tissues.Our study provided the first comprehensive profile as well as the alteration of modifications in different RNA species in thyroid carcinoma and matched tumor-adjacent normal tissues.The altered pattern RNA modifications may serve as the indicator of thyroid carcinoma.Moreover,this study may promote the in-depth understanding of the regulatory roles of RNA modifications in thyroid carcinoma.

Transformation of 5-Carboxylcytosine to Cytosine Through C-C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Active demethylation of 5-methylcytosine(5mC)can be realized through ten-eleven translocation(TET)dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC),and 5-carboxylcytosine(5caC),followed by thymine DNA glycosylase(TDG)-initiated base excision repair(BER).The TDG-BER pathwaymay lead to the generation of DNA strand breaks,potentially compromising genome integrity.Alternatively,direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks.However,cleavage of the C–C bond in 5caC in human cells remains an open question.We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate.After incubation with whole-cell protein extract or transfection into human cells,we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography–tandem mass spectrometry(LCMS/MS)analyses at both the mononucleotide and oligodeoxynucleotide levels.Our results clearly showed the direct conversion of 5caC to cytosine in human cells,providing evidence to support a novel pathway for active DNA demethylation.