Our understanding of the functional role of posttranscriptionally modified nucleosides in protein synthesis, especially those localized to ribosomal ribonucleic acids (rRNAs), is limited largely by the lack of methods that can identify and characterize their interactions with proteins. The long-term goal of our research has been and continues to be to develop appropriate mass spectrometric approaches to characterize the structure of the ribosome in terms of RNA and RNA-protein interactions. The goals of this renewal are to develop new and improved mass spectrometry (MS) approaches to identify and characterize RNA-protein interactions within the ribosome, and to apply these methods to obtain biologically relevant information about ribosome structure and function.
The first aim will focus on cross-link identification and sequencing. Those developments will be used to identify specific recognition elements of the RNAs and ribosomal proteins involved in the initiation step in protein synthesis.
The second aim will focus on identifying and quantifying pseudouridine. Those developments will be used to quantitatively characterize pseudouridines present in 23S rRNA. The significance of these proposed studies include bioanalytical MS developments for identifying and sequencing RNA-protein (or DNA-protein) cross-links and the quantitative determination of pseudouridine with site specificity. This research plan will yield important biological information on the ribosome including how messages lacking the Shine-Dalgarno motif are bound before translation, the role of ribosomal protein L31 in the initiation step, and the mode of action for the 23S rRNA pseudouridine synthase RluD. Innovations in the proposed research plan include developing liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) for selective identification of nucleic acid-protein cross-links, the use of electron transfer dissocation (ETD) for sequencing the peptide moiety in an oligonucleotide:peptide heteroconjugate, which leads to a robust MS/MS method for cross-link sequencing, and creating a quantitative assay for pseudouridine. This research plan will yield new and improved approaches for characterizing RNA-protein interactions. Additionally, while the technology and method developments from this research will be used to characterize the ribosome, many of the developments will have broad applicability for investigators interested in characterizing other ribonucleoprotein complexes, DNA-protein complexes, or other nucleic acid systems. Importantly, this research plan will provide new tools and information of importance about protein synthesis, which is fundamentally significant and relevant to human health.

Public Health Relevance

Protein synthesis is a fundamental biological process required for life. The site of protein synthesis, the ribosome, is a large macromolecule composed of RNAs and proteins. Understanding how RNAs and proteins interact, and how they are modified, is important to differentiate bacterial ribosomes from those of higher organisms, such as mammals. This research will create new tools that enable researchers to characterize ribosomes, and these tools will be used here to obtain important information relating to early steps in protein synthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058843-11
Application #
8296558
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
1999-02-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
11
Fiscal Year
2012
Total Cost
$244,099
Indirect Cost
$84,716
Name
University of Cincinnati
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Addepalli, Balasubrahmanyam; Limbach, Patrick A (2016) Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF. J Biol Chem 291:22327-22337
Ross, Robert; Cao, Xiaoyu; Yu, Ningxi et al. (2016) Sequence mapping of transfer RNA chemical modifications by liquid chromatography tandem mass spectrometry. Methods 107:73-8
Wetzel, Collin; Limbach, Patrick A (2016) Mass spectrometry of modified RNAs: recent developments. Analyst 141:16-23
Addepalli, Balasubrahmanym; Lesner, Nicholas P; Limbach, Patrick A (2015) Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia. RNA 21:1746-56
Sample, Paul J; Kořený, Luděk; Paris, Zdeněk et al. (2015) A common tRNA modification at an unusual location: the discovery of wyosine biosynthesis in mitochondria. Nucleic Acids Res 43:4262-73
Sample, Paul J; Gaston, Kirk W; Alfonzo, Juan D et al. (2015) RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids. Nucleic Acids Res 43:e64
Cao, Xiaoyu; Limbach, Patrick A (2015) Enhanced detection of post-transcriptional modifications using a mass-exclusion list strategy for RNA modification mapping by LC-MS/MS. Anal Chem 87:8433-40
Dator, Romel P; Gaston, Kirk W; Limbach, Patrick A (2014) Multiple enzymatic digestions and ion mobility separation improve quantification of bacterial ribosomal proteins by data independent acquisition liquid chromatography-mass spectrometry. Anal Chem 86:4264-70
Puri, Pranav; Wetzel, Collin; Saffert, Paul et al. (2014) Systematic identification of tRNAome and its dynamics in Lactococcus lactis. Mol Microbiol 93:944-56
Köhrer, Caroline; Mandal, Debabrata; Gaston, Kirk W et al. (2014) Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile. Nucleic Acids Res 42:1904-15

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