The proposed research focuses on mRNA pseudouridylation and its effect on coding. Pseudouridine (?) is the most abundantly modified nucleotide, which is distinct from all other known nucleotides. Our recent work indicates that ?, when introduced into nonsense and sense codons, results in coding specificity changes. Our preliminary results also suggest that mRNA contains naturally-occurring ?s. We are now in a unique position to identify amino acids and tRNAs that are coded for by pseudouridylated codons, thereby dramatically expanding the current genetic code.
Three specific aims are proposed.
Aim 1 : To determine specific amino acids encoded by specific pseudouridylated sense codons Encouraged by our initial identification of ?-mediated coding specificity changes, we propose to use in vivo and in vitro translation coupled with mass spectrometry (MS) sequencing to define an amino acid code encompassing all pseudouridylated sense codons. We will work towards improving pseudouridylation efficiency in yeast, thus enhancing the production of proteins translated from pseudouridylated mRNAs and facilitating MS sequencing. If necessary, we will carry out the experiments in the Xenopus oocyte injection and the wheat embryo in vitro translation systems, in which pseudouridylated mRNA can be directly used for translation. We believe that through the use of these systems, a complete amino acid code will emerge.
Aim 2 : To identify specific tRNA species (anticodons) that recognize pseudouridylated codons. Although the MS sequencing approach will reveal the genetic code at the amino acid level, the question of which member(s) of the tRNA isoacceptor families, deduced from the amino acid code, is responsible for the decoding of a pseudouridylated codon remains unanswered. We propose to use two independent approaches, namely, tRNA over-expression and tRNA-ribosome-codon filter-binding, to decode, at the tRNA level, each pseudouridylated codon. In doing so, we will be able to fully decipher the genetic code of pseudouridylated codons. The identification of specific tRNA species will further allow a collaborative effort to crystallize the ribosomes that contain pseudouridylated codons and their newly-identified cognate tRNAs. Such analyses will provide a more complete understanding of how pseudouridylated codons are recognized.
Aim 3 : To identify naturally occurring ?s in mRNAs and natural alternative coding in cells. Our preliminary experiments strongly suggest that pseudouridylation occurs naturally in mRNAs. We believe that, with the help of modern technology--deep sequencing, it is time to conduct an unbiased global search for ?s in mRNAs. Specifically, we will combine CMC-modification-primer-extension and CLIP with deep sequencing to globally screen all mRNAs for naturally occurring ? content. ?s thus identified will then be verified and quantified by recently developed site-specific pseudouridylation assays. The identification of naturally occurring ?s will in turn allow us to identify naturally occurring alternative coding.

Public Health Relevance

In this application, we propose to investigate how pseudouridylation of codons affects coding during translation and how widespread naturally occurring pseudouridine is in mRNAs. There is no doubt that completion of the work will greatly enhance our understanding of the roles of RNA modifications in codon recognition, and will dramatically expand the current genetic code. From the clinical perspective, a large number of human genetic diseases are associated with codon mutations, especially those that create a premature nonsense codon. Our preliminary results indicate that targeted pseudouridylation at premature nonsense codons specifically suppresses premature translation termination, thus restoring the production of functional protein. Our application is thus of clinical relevance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM104077-01
Application #
8419298
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2012-09-24
Project End
2016-08-31
Budget Start
2012-09-24
Budget End
2013-08-31
Support Year
1
Fiscal Year
2012
Total Cost
$291,650
Indirect Cost
$101,650
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Zhao, Yang; Dunker, William; Yu, Yi-Tao et al. (2018) The Role of Noncoding RNA Pseudouridylation in Nuclear Gene Expression Events. Front Bioeng Biotechnol 6:8
Adachi, Hironori; De Zoysa, Meemanage D; Yu, Yi-Tao (2018) Post-transcriptional pseudouridylation in mRNA as well as in some major types of noncoding RNAs. Biochim Biophys Acta Gene Regul Mech :
De Zoysa, Meemanage D; Wu, Guowei; Katz, Raviv et al. (2018) Guide-substrate base-pairing requirement for box H/ACA RNA-guided RNA pseudouridylation. RNA 24:1106-1117
Ge, Junhui; Karijolich, John; Zhai, Yingzhen et al. (2017) 5-Fluorouracil Treatment Alters the Efficiency of Translational Recoding. Genes (Basel) 8:
De Zoysa, Meemanage D; Yu, Yi-Tao (2017) Posttranscriptional RNA Pseudouridylation. Enzymes 41:151-167
Poole, Aaron R; Vicino, Ian; Adachi, Hironori et al. (2017) Regulatory RNPs: a novel class of ribonucleoproteins that potentially contribute to ribosome heterogeneity. Biol Open 6:1342-1354
Xu, Chao; Ishikawa, Hideaki; Izumikawa, Keiichi et al. (2016) Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly. Genes Dev 30:2376-2390
Huang, Chao; Karijolich, John; Yu, Yi-Tao (2016) Detection and quantification of RNA 2'-O-methylation and pseudouridylation. Methods 103:68-76
Wu, Guowei; Radwan, Mohamed K; Xiao, Mu et al. (2016) The TOR signaling pathway regulates starvation-induced pseudouridylation of yeast U2 snRNA. RNA 22:1146-52
Huang, Chao; Wu, Guowei; Yu, Yi-Tao (2016) Purification and Functional Reconstitution of Box H/ACA Ribonucleoprotein Particles. Methods Mol Biol 1421:97-109

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