skip to main content


The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Friday, May 17 until 8:00 AM ET on Saturday, May 18 due to maintenance. We apologize for the inconvenience.

Title: 5-Halogenation of Uridine Suppresses Protonation-Induced Tautomerization and Enhances Glycosidic Bond Stability of Protonated Uridine: Investigations via IRMPD Action Spectroscopy, ER-CID Experiments, and Theoretical Calculations
Award ID(s):
1709789 1357887 0922819
Author(s) / Creator(s):
; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Journal of the American Society for Mass Spectrometry
Page Range / eLocation ID:
2165 to 2180
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Chronic inflammation associated with inflammatory bowel disease (IBD) results in increased oxidative stress that damages the colonic microenvironment. A low level of serum bilirubin, an endogenous antioxidant, has been associated with increased risk for Crohn's disease (CD), but no study has tested another common IBD ulcerative colitis (UC). Bilirubin is metabolized in the liver by uridine glucuronosyltransferase 1A1 (UGT1A1) exclusively. Genetic variants cause functional changes in UGT1A1 which result in hyperbilirubinemia, which can be toxic to tissues if untreated and results in a characteristic jaundiced appearance. Approximately 10% of the Caucasian population is homozygous for the microsatellite polymorphism UGT1A1*28, which results in increased total serum bilirubin levels due to reduced transcriptional efficiency of UGT1A1 and an overall 70% reduction in UGT1A1 enzymatic activity. The aim of this study was to examine whether bilirubin levels are associated with the risk for ulcerative colitis (UC). Using the Informatics for Integrating Biology and the Bedside (i2b2), a large case-control population was identified from a single tertiary care center, Penn State Hershey Medical Center (PSU). Similarly, a validation cohort was identified at Virginia Commonwealth University Medical Center. Logistic regression analysis was performed to determine the risk of developing UC with lower concentrations of serum bilirubin. From the PSU cohort, a subset of terminal ileum tissue was obtained at the time of surgical resection to analyze UGT1A1 gene expression (which encodes the enzyme responsible for bilirubin metabolism). Similar to CD patients, UC patients also demonstrated reduced levels of total serum bilirubin. Upon segregating serum bilirubin levels into quartiles, risk of UC increased with reduced concentrations of serum bilirubin. These results were confirmed in our validation cohort. UGT1A1 gene expression was up-regulated in the terminal ileum of a subset of UC patients. Lower levels of the antioxidant bilirubin may reduce the capability of UC patients to remove reactive oxygen species leading to an increase in intestinal injury. One potential explanation for these lower bilirubin levels may be up-regulation of UGT1A1 gene expression, which encodes the only enzyme involved in conjugating bilirubin. Therapeutics that reduce oxidative stress may be beneficial for these patients. 
    more » « less
  2. Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N 2-methylguanosine, N 2, N 2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N 2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context. 
    more » « less