ABSTRACT
Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.
Subject(s)
Epigenesis, Genetic , Gene Library , High-Throughput Nucleotide Sequencing/methods , RNA/genetics , Transcriptome , Calibration , Gene Expression Regulation , HeLa Cells , HumansABSTRACT
Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.
