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
Puri, Pranav; Wetzel, Collin; Saffert, Paul et al. (2014) Systematic identification of tRNAome and its dynamics in Lactococcus lactis. Mol Microbiol 93:944-56
Frankel, Laurie K; Sallans, Larry; Limbach, Patrick A et al. (2013) Oxidized amino acid residues in the vicinity of Q(A) and Pheo(D1) of the photosystem II reaction center: putative generation sites of reducing-side reactive oxygen species. PLoS One 8:e58042
Frankel, Laurie K; Sallans, Larry; Bellamy, Henry et al. (2013) Radiolytic mapping of solvent-contact surfaces in Photosystem II of higher plants: experimental identification of putative water channels within the photosystem. J Biol Chem 288:23565-72
Russell, Susan P; Limbach, Patrick A (2013) Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC-UV-MS. J Chromatogr B Analyt Technol Biomed Life Sci 923-924:74-82
Nayak, Nihar R; Putnam, Andrea A; Addepalli, Balasubrahmanyam et al. (2013) An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE. Plant Cell 25:2573-86
Catron, Brittany; Caruso, Joseph A; Limbach, Patrick A (2012) Selective detection of peptide-oligonucleotide heteroconjugates utilizing capillary HPLC-ICPMS. J Am Soc Mass Spectrom 23:1053-61
Anton, Brian P; Russell, Susan P; Vertrees, Jason et al. (2010) Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis. Nucleic Acids Res 38:6195-205
Durairaj, Anita; Limbach, Patrick A (2008) Matrix-assisted laser desorption/ionization mass spectrometry screening for pseudouridine in mixtures of small RNAs by chemical derivatization, RNase digestion and signature products. Rapid Commun Mass Spectrom 22:3727-34
Pourshahian, Soheil; Limbach, Patrick A (2008) Application of fractional mass for the identification of peptide-oligonucleotide cross-links by mass spectrometry. J Mass Spectrom 43:1081-8
Brock, Jay E; Pourshahian, Soheil; Giliberti, Jacqueline et al. (2008) Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG. RNA 14:2159-69

Showing the most recent 10 out of 26 publications