INVESTIGATE SEQUENCE SPECIFICITY IN THE BIOSYNTHESIS AND RECOGNITION OF RNA CHEMICAL
MODIFICATIONS
PROJECT SUMMARY
Chemical modifications are prevalent in the cellular RNA across all domains of life; they expand the chemical
space beyond the four natural building blocks of RNA, modulate folding, intermolecular interactions involving
RNA, and regulate gene expression. Growing evidence shows that several RNA chemical modifications can be
reversed by endogenous enzymes and can respond to external cues, including metabolic signaling, nutrient
starvation, oxidative stress, and temperature change. Interestingly, many proteins known to directly or indirectly
regulate RNA chemical modifications are found to be dysregulated in physiological defects or diseases, forming
the basis of many exciting hypotheses to discover the role of the epitranscriptome in gene expression regulation.
While our understanding and therapeutic exploitation of epitranscriptome-based gene expression control
continue to expand, the molecular mechanisms that govern this regulation remain poorly understood. A critical
question in the epitranscriptome field is the sequence specificity of various RNA modifications: how they are
installed on specific sequence locations and how they regulate specific protein-RNA recognition. Studying the
sequence contexts of chemically modified endogenous RNA has been technically challenging. With recent
advances in high-throughput sequencing-based technologies, we can now map and quantify only a handful of
RNA chemical modifications (out of over 150 types) in their native sequence contexts inside cells. The pilot
mapping studies revealed conserved sequence elements associated with the occurrence of specific
modifications across different domains of life. That such conserved occurrence of modifications, rather than
being randomly distributed, strongly suggests the involvement of dedicated endogenous machinery that
regulates modifications with high specificity. However, we do not understand why and how the modifications are
installed at specific locations and regulate biology, most likely in a sequence-dependent manner. To address
this knowledge gap, we need methods to detect RNA modifications confidently within sequence contexts that
offer high accuracy and throughput. Here we propose a program focusing on developing such methods and
performing systematic biochemical characterization of the sequence specificity of effector proteins by combining
in vitro high-throughput assay and in cellulo massive parallel reporter assay approaches. Inspired by recent
findings that modification reader proteins may recognize more than one chemical modification, we aim to revisit
the molecular recognition mechanism of the most heavily modified RNA – transfer RNAs in human cells.
By developing more advanced detection tools in mapping RNA chemical modifications in biological RNA and
designed oligonucleotide libraries modified in vitro or in cellular reporter assays, we will elucidate the
fundamental biochemical properties of the critical regulators of the epitranscriptome with unprecedented
efficiency and comprehensiveness, leading to a better understanding of RNA regulation, and new opportunities
to exploit and control the epitranscriptome.
