Towards site-specific functional analysis of RNA N6-methyladenosine and 5-methylcytosine in Arabidopsis thaliana

Abstract

Phenotype prediction from DNA or RNA sequence in eukaryotes is difficult as a result of the multiple layers of gene expression regulation. One of these regulatory layers is RNA modification which occurs either co-transcriptionally or post-transcriptionally to RNA and affects many aspects of RNA biology. Recent transcriptome-wide insights of RNA modifications have begun to elucidate the extent of this landscape, leading to the proposition of the “epitranscriptome”. However, understanding the “epitranscriptome” and its consequences to RNA metabolism, functional relevance and mechanism of action remains an enormous undertaking. The lack of tools to specifically manipulate an RNA modification has hampered the un-biased evaluation of their importance as well as understanding of the mechanism underlying their activities in a site-specific manner. During my research, I investigated the RNA-guided RNA targeting system CRISPRdCas13 and the RNA-based technique Short Tandem Target Mimic (STTM) regarding their potential to develop programmable systems for targeted demethylation of two RNA modifications N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in Arabidopsis thaliana. Initial results indicated unsuccessful interference to m5C deposition using the STTM approach. In contrast, fusion of dCas13 to RNA modifying domains ALKBH10B or human TEN-ELEVEN TRANSLOCATION1 (hTET1) respectively enabled alterations of m6A- or m5C-bearing reporter or endogenous transcripts in either Nicotiana benthamiana or A. thaliana. However, further investigation is required to understand the robustness of both approaches. Possible improvements are discussed for targeted RNA demethylation tools using CRISPRdCas13. In addition, the functional relevance of RNA m5C in root development in A. thaliana was also extensively investigated with regards to previously proposed involvement of two transcripts SHORT HYPOCOTYL 2 (SHY2/IAA3) and INDOLEACETIC ACID-INDUCED PROTEIN 16 (IAA16). Phenotypic and mRNA quantification analyses of m5C-deficient and SHY2/IAA3 loss-of-function mutants refuted a key role for SHY2/IAA3 and IAA16 in m5C regulation of root development. Although I identified an m5C site C348 on SHY2/IAA3 using bisulfite RNA amplicon sequencing and initial nucleotide conservation analysis suggested that this site is more conserved than other non-methylated cytosines within SHY2/IAA3 mRNA, functional analysis using dCas13-hTET1 conjugate did not support the importance of this C348 in planta. In summary, RNA-guided CRISPR-dCas13 and multi-strategy approaches for site-specific functional study offers potential to significantly improve our understanding of RNA modifications.