Epitranscriptomics and the emerging roles of

RNA modifications in RNA function

While “GENE” and “GENE” both spell the same, formatting of the latter word changes the emphasis we place on it. This is analogous to the study of epitranscriptomics, where the underlying RNA sequence can be the same but chemical RNA modifications (epitranscriptome) change how the cell regulates and expresses the RNA. This is why epitranscriptomics research has revealed new regulatory pathways that could not be determined from simply sequencing the transcriptome. To date, research has demonstrated that the epitranscriptome regulates almost all forms of known RNA processing pathways. Consequently, changes in the epitranscriptome or misregulation of epitranscriptomic factors contribute to the progression of a multitude of diseases. Therapeutics that target epitranscriptomic factors, which include “writers” and “erasers” that respectively add or remove the N6-methyladenosine (m6A) modification as well as “readers” that target m6A, are already scheduled for clinical trials in 2021. 

The goal of our laboratory is to discover new RNA modifications and epitranscriptomic factors as well as determine their mechanisms for regulating molecular and cellular processes and how these ultimately impact the progression of various diseases.


1) Develop technologies for high-resolution epitranscriptome sequencing

The invention of single-base-resolution RNA modification (epitranscriptome) sequencing has revealed great insights into epitranscriptomic regulation of cellular processes. For instance, our lab developed m6A-crosslinking-exonuclease (m6ACE) sequencing for quantitative single-base-resolution mapping of transcriptome-wide RNA methylation profiles.  Using m6ACE, we generated the first ever human atlas of RNA methylomes distinct to each and every RNA writer or eraser known thus far. Insights garnered from this atlas allowed us to challenge the notion that erasers mediate cycles of methylation reversal and furthermore, thereby redefining their functional roles as suppressors of aberrant RNA methylation (Koh et al. 2019). Therefore, this highlights the importance of resolution in epitranscriptome sequencing. 

Given that there are many other RNA modifications that still lack a precise mapping method, we are interested in developing more tools to sequence other RNA modifications of interest at single-base resolution.

Another form of high-resolution sequencing is single-cell-resolution. Just as how single-cell transcriptome-sequencing has revealed new cellular subtypes that are stratified by their gene expression signatures, mapping single-cell epitranscriptomes can likewise reveal new cellular subtypes that are stratified by specific RNA modifications. We are currently exploring techniques that will facilitate single-cell epitranscriptome sequencing. For instance, we have recently explored antibody-independent methods for sequencing RNA modifications. By harnessing the quantitative precision of m6ACE, we generated training datasets for developing a new algorithm, xPore to map and quantify m6A methylation using direct RNA sequencing, generating transcriptome-wide and single-base-resolution RNA methylomes (Pratanwanich et al. 2020).

Relevant publications:

Pratanwanich PN*, Yao F, Chen Y, Koh CWQ, Wan YK, Hendra C, Poon P, Goh YT, Yap PML, Yuan CJ, Chng WJ, Ng SB, Thiery A, Goh WSS*, Göke J*. Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore (2021) Nature Biotechnology.

Koh CWQ, Goh YT, Goh WSS*. Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes (2019) Nature Communications.

2) Identify and functionally characterize novel epitranscriptomic factors

RNA is modified by catalytic Writers that often function in a complex with co-factors guides, which direct Writers to modify specific RNA sequences. Identification of these Writers and co-factors is required for generating the knockout (KO) cell lines that form the basis of necessary tools for studying the function of the associated RNA modification. In our recent work, we screened for candidate RNA methylation Writers and used m6ACE to demonstrate that Mettl4 is a novel N6-methyl-2’O-methyl-adenosine (m6Am) Writer. By generating Mettl4-KO cells, we demonstrated that Mettl4 absence causes loss of methylation on U2 snRNA, leading to a global change in pre-mRNA splicing patterns (Goh et al. 2020). Following up on this, we will explore other novel epitranscriptomic Writer/co-factor candidates and characterize the function of their associated RNA modification. 

We were also one of the first groups to identify Pcif1 as a novel writer for TSS-m6Am by using m6ACE to map differential RNA methylomes between wildtype and Pcif1-ko cells at the TSS (Koh et al. 2019).

Pcif1 deletion causes loss in RNA methylation signals specific to the TSS but not the 3’end of genes.

RNA modifications also exert their regulation by recruiting specific RNA-modification-binding Readers. The function of the RNA modifications can thus be inferred from the function of the identified Reader. To that end, we plan to utilize quantitative proteomics and biochemical tools to identify and mechanistically characterize novel epitranscriptomic Readers.

Relevant publications:

Goh YT, Koh CWQ, Sim DY, Roca X*, Goh WSS*. METTL4 catalyzes N6-methylation of adenosines in U2 snRNA to regulate pre-mRNA splicing (2020) Nucleic Acids Research.

Koh CWQ, Goh YT, Goh WSS*. Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes (2019) Nature Communications.

3) Investigate the roles of epitransciptomics in disease progression

Dysregulation of epitranscriptomic factors has been shown to be an underlying cause of various diseases. For instance, the m6A Writer Mettl3 is down-regulated in various cancers and as a result, alters gene expression profiles in these cancers and contributes to cancer phenotypes. Despite the fact that Mettl3 is similarly down-regulated in all these cancers, the resultant genes that exhibit reduced m6A methylation are uniquely different in each cancer. This highlights the importance of being able to precisely map differential methylation patterns so as to identify mis-modified genes and characterize how their aberrant RNA modifications contribute to disease phenotypes. Therefore, our ongoing work utilizes the quantitative precision of m6ACE to accurately profile differential RNA methylomes between tumour cells and matched healthy cells derived from the same individual colorectal cancer patient. This will allow us to assess how tumour-specific RNA methylomes contribute to colorectal cancer progression within an isogenic setting.

We will eventually incorporate the other novel sequencing technologies that we are developing so as to expand this research angle to investigate the role of epitranscriptomics in other diseases.