Expanding world of epitranscriptome

Higher-order biological processes, including development, differentiation and complex mental activity, are the result of sophisticated regulation of gene expression. Dysregulation of gene expression often causes a variety of human diseases. Thus, a thorough investigation of the mechanism by which gene expression is regulated is critical for understanding higher-order biological processes, and will enable us to develop effective therapeutic interventions.
RNA molecules contain various types of post-transcriptional modifications that are required for modulating RNA function and gene expression. In fact, over 130 different RNA modifications have been identified in RNA molecules across all domains of life. Compared to the limited variation of DNA modifications, a wide variety of RNA modifications appear to be a strategy by which RNA molecules acquire a greater variety of biological functions. Since RNA modifications are regulated by the expression levels of RNA-modifying enzymes and/or cellular concentrations of substrate metabolites, levels of RNA modifications are altered in a spatiotemporal manner under various cellular conditions during development and differentiation, and by exposure to environmental stresses. Moreover, we found the first instance of human disease caused by lack of RNA modification, and proposed "RNA modopathy" as a new category of human disease. We are tackling to elucidate various biological phenomena associated with RNA functions.

RNA modifications associated with various biological functions

RNA molecules are frequently modified post-transcriptionally and these modifications are required for proper RNA functions. To date, over 130 different type of chemical modifications have been identified in various RNA molecules across all domains of life. They have a wide variety of chemical diversity including methylation, hydroxylation, acetylation, deamination, isomerization, selenylation, reduction, cyclization, and conjugation with amino acids and sugars. The modifications that occur in RNA molecules stabilize tertiary structures, modulate affinity for RNA-binding proteins, regulate decoding of genetic codes, and determine the subcellular localization and lifetime of RNAs. However, the exact function and biogenesis of many of these modifications have yet to be determined. We are studying various biological processes associated with RNA modifications through discovery of novel RNA modifications and RNA-modifying enzymes. So far, we reported several novel modifications, and over 40 RNA-modifying enzymes.

Decoding of genetic information and protein synthesis

Genetic information in DNA is transcribed to mRNA, and then translated to protein on the ribosome. In general, fidelity of translation is estimated in the range of 10-4 to 10-5 per codon. Living organisms have various measures to maintain accurate protein synthesis. mRNA codons are recognized by tRNA anticodons at the ribosomal A-site to be converted to corresponding aminoacids. A wide variety of modifications found in the anticodon region of tRNAs play critical roles in precise decoding in translation. We are exploring novel tRNA modifications required for accurate decoding, and studying molecular function and physiological significance of these modifications. At the early stage of translation, peptidyl-tRNAs are frequently dissociated from the ribosome (pep-tRNA drop off). We have clear evidence showing that this event contributes to quality control of protein synthesis. We are exploring novel mechanisms to maintain accurate protein synthesis in the cell using genetics and biochemistry.

Epitranscriptome and biological functions

The 5' cap structure has been a characteristic post-transcriptional modification in eukaryotic mRNAs and non-coding (nc)RNAs, and well studies for many years. However, recent advances in analytical methods using next generation sequencing (NGS) technologies have detected several other RNA modifications in mRNAs and ncRNAs, such as inosines (I), 5-methylcytidines (m5C), N6-methyladenosines (m6A), pseudouridine (Ψ) and 1-methyladenosine (m1A). These findings stress the importance of RNA modifications as regulatory elements in gene expression, and the process based on RNA modification is also referred to as "epitranscriptome". To explore RNA modifications in transcriptome-wide manner, we have been carrying out a project to develop mapping methods for RNA modifications based on RNA chemical biology assisted by NGS technologies. For identification of A-to-I editing sites, we developed a biochemical method for detecting inosines, called ICE-seq, and successfully mapped over 30,000 novel inosine sites in the human brain transcriptome. We are now investigating functional roles of these I sites as regulatory elements in gene expression.

Molecular pathogenesis of RNA modopathy

It is a critical basic research to understand pathogenesis of human disease at molecular level for the purpose of development of effective therapeutic measures and diagnostic techniques. Mitochondrial encephalomyopathies are severe human diseases caused by mitochondrial dysfunction. We previously revealed that lack of tRNA modification is a primary cause of mitochondrial diseases. This is the first confirmation of a human diseases caused by an RNA modification disorder. Thus, we are proposing "RNA modopathy" as a new category of human diseases. We are striving to reveal molecular pathogenesis of various RNA modopathies through multidimensional approaches using clinical specimens, patient tissues and cells, and knockout mice.

RCC and RNA-MS: platform technologies for RNA analysis

To analyze complicated RNA modifications and terminal chemical structures, we have developed methodologies that allow us to examine RNA as a chemical molecule rather than a simple nucleotide sequence. Isolation of individual RNA molecules is not general, rather it is a highly skillful and challenging technique that requires large amounts of specimens, complicated and laborious procedures, and a great deal of experience. We developed our original method called "reciprocal circulating chromatography (RCC)" to isolate multiple species of RNAs in parallel, and constructed an RCC machine that enables us to perform automated RNA isolation. Using this method, we successfully isolated various types of individual RNAs including miRNAs and mRNAs. Furthermore, to analyze the isolated RNA molecules, we developed a highly sensitive detection system for RNA molecules based on mass spectrometry (RNA-MS). We also developed a series of related technologies for RNA-MS including pretreating the RNA molecules to be analyzed, a specialized capillary liquid chromatography method to separate RNA fragments that are limited in quantity, optimization of mass spectrometry to maximize ionization and detection efficiency, an algorithm to calculate complex mass spectra, and software to analyze RNA sequence from product ions generated by collision-induced dissociation (CID). RNA-MS has been established by combining all of these technical innovations. In fact, we can successfully analyze as little as 50 attomoles of an RNA molecule, which represents a 106-fold increase in the sensitivity of detection over the past 10 years. With this sensitivity, our method can be widely applied at the practical level. The development of RNA-MS has enabled us to discover several novel modified RNA bases and dozens of RNA-modifying enzymes.


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