Existence of Diverse Modifications in Small-RNA Species Composed of 16-28†Nucleotides.

RNA contains diverse modifications that exert an important influence in a variety of cellular processes. So far, more than 150 modifications have been identified in various RNA species, mainly in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). In contrast to rRNA, tRNA, and mRNA, the known modifications in small RNA species have been primarily limited to 2'-O-ribose methylation in plants and inosine in mammals. The methylation of small RNAs in mammals is still unclear. Current methods widely used in the characterization of small RNAs are mainly based on the strategy of nucleic acid hybridization and sequencing, which cannot characterize modifications in small RNAs. Herein, we have systematically investigated modifications in small RNAs composed of 16-28 nucleotides (nt) by establishing an effective isolation and neutral enzymatic digestion of small RNAs in combination with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This method allowed us to simultaneously detect 57 different types of nucleoside modification. By using this approach, we revealed 24 modifications in small RNAs comprising 16-28 nt from human cells. In addition, we found that the obesity-associated protein (FTO) may demethylate N6 -methyladenosine (m6 A) and N6 ,2'-O-dimethyladenosine (m6 Am) in small RNAs of 16-28 nt. Our study demonstrates the existence of diverse modifications in small RNAs composed of 16-28 nt, which may promote in-depth understanding of the regulatory roles of noncoding RNAs.

Existence of Internal N7-Methylguanosine Modification in mRNA Determined by Differential Enzyme Treatment Coupled with Mass Spectrometry Analysis.

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.

Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two folds in both gastric cancer and lymphoma; one ribose conjugate increased more than two folds in both esophagus cancer and lymphoma. The developed analytical method provides a good platform to study the modified ribonucleosides in human body fluids.

Formation and determination of the oxidation products of 5-methylcytosine in RNA.

Similar to the reversible epigenetic modifications on DNA, dynamic RNA modifications were recently considered to constitute another realm for biological regulation in the form of "RNA epigenetics". 5-Methylcytosine (5-mC) has long been known to be present in RNA from all three kingdoms of life. However, the functions of 5-mC in RNA have not been fully understood, especially for the RNA demethylation mechanism. The discovery of 5-hydroxymethylcytosine (5-hmC) in RNA together with our recently reported 5-formylcytosine (5-foC) in RNA indicated that 5-mC in RNA may undergo the same cytosine oxidation demethylation pathway with generating intermediates 5-hmC, 5-foC, and 5-carboxylcytosine (5-caC) by ten-eleven translocation (Tet) proteins as that in DNA. However, endogenous 5-caC in RNA has not been observed so far. In the current study, we established a method using chemical labeling coupled with liquid chromatography-mass spectrometry analysis for the sensitive and simultaneous determination of the oxidative products of 5-mC. Our results demonstrated that the detection sensitivities of 5-mC, 5-hmC, 5-foC and 5-caC in RNA increased by 70-313 folds upon 2-bromo-1-(4-diethylaminophenyl)-ethanone (BDEPE) labeling. Using this method, we discovered the existence of 5-caC in the RNA of mammals. In addition, we found the 5-mC occurs in all RNA species including mRNA, 28S rRNA, 18S rRNA and small RNA (<200 nt). However, 5-hmC, 5-foC and 5-caC mainly occur in mRNA, and barely detected in other types of RNA. Furthermore, we found that the content of 5-hmC in the RNA of human colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) tissues significantly decreased compared to tumor adjacent normal tissues, suggesting that 5-hmC in RNA may play certain functional roles in the regulation of cancer development and formation.

DNA hydroxymethylation age of human blood determined by capillary hydrophilic-interaction liquid chromatography/mass spectrometry.

BACKGROUND: Aging is a complex phenomenon and characterized by a progressive decline in physiology and function of adult tissues. However, it hasn't been well established of the correlation between aging and global DNA methylation and hydroxymethylation that regulate the growth and development of higher organisms. RESULTS: We developed an on-line trapping/capillary hydrophilic-interaction liquid chromatography/electrospray ionization-mass spectrometry method for ultra-sensitive and simultaneous quantification of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in genomic DNA from human blood. Limits of detection for 5-mC and 5-hmC were 0.04 and 0.13 fmol, respectively. The imprecision and recovery of the method were determined with the relative standard deviations (RSDs) and relative errors being <11.2 and 14.0 %, respectively. We analyzed the contents of 5-mC and 5-hmC in genomic DNA of blood from 238 healthy people aged from 1 to 82 years. The results showed that 5-hmC content was significantly decreased and highly correlated with aging process, while 5-mC only showed slight correlation with age. We then established a DNA hydroxymethylation age model according to 5-hmC content with a mean absolute deviation (MAD) of approximate 8.9 years. We also calculated the mean relative error (MRE) using the predicted ages based on the age model and the chronological ages. The results showed that the MRE was 18.3 % for samples with ages from 20 to 82 years (95 % confidence interval, N = 190). CONCLUSIONS: The global DNA hydroxymethylation represents a strong and reproducible mark of chronological age, which could be potentially applied in health assessment and prevention of diseases. The identification of biological or environmental factors that influence DNA hydroxymethylation aging rate may permit quantitative assessments of their impacts on health.