Decoding the Mysteries of RNA Methylation: Exploring Bisulfite Sequencing

Understanding the mechanisms regulating gene expression is crucial in the fascinating world of genetics. The addition of chemical modifications to RNA molecules is known as RNA methylation. Bisulfite sequencing, initially used for DNA methylation analysis, is a powerful technique for deciphering the methylation patterns. In this article, we will explore bisulfite sequencing for RNA methylation research, explaining its principles, applications, and insights it provides into the complex world of epigenetics (epitranscriptomics).

Understanding RNA methylation

Before we dive into bisulfite sequencing, let's briefly explore RNA methylation. Hundreds of different RNA modifications have been discovered. Some of the most studied modifications are N6-methyladenosine (m6A), pseudouridine and 5-methylcytidine (m5C). The latter describes adding a methyl group to the cytosine C5 atom.

Methylation Patterns and Gene Regulation

Methylation patterns profoundly influence gene expression. Hypermethylation, which involves adding methyl groups, often occurs in CpG islands near gene promoters. This dense methylation can inhibit transcription factors from binding to DNA, resulting in gene silencing. In contrast, hypomethylation, or removing methyl groups, is associated with increased gene expression. By mapping and understanding these patterns, researchers can gain insights into regulating specific genes and their involvement in various diseases. 

Understanding Bisulfite Sequencing

Bisulfite sequencing is a technique for analysing the methylation status of RNA molecules in m5C (and, to a lesser extent, m4C).

Step 1. Bisulfite Treatment:

Bisulfite treatment is the core step in bisulfite sequencing. Sodium bisulfite selectively converts unmethylated cytosine residues to uracil, while methylated cytosines remain unaltered. The reaction is carried out under controlled conditions to ensure the conversion is specific and minimal degradation of RNA occurs.

Step 2. Reverse Transcription:

Following bisulfite treatment, the converted RNA is subjected to reverse transcription, where uracil is read as thymine by the reverse transcriptase enzyme. This step converts all the uracils into thymines, while the methylated cytosines are retained as cytosines in the cDNA.

Step 3. Sanger or Next Generation Sequencing:

The cDNA generated through reverse transcription is then subjected to Sanger sequencing or high-throughput NGS sequencing. By comparing the sequencing results to a reference genome or transcriptome, researchers can identify the positions of methylated cytosines, providing insights into the RNA methylation landscape.

Applications of Bisulfite Sequencing for RNA Methylation 

Bisulfite sequencing has revolutionised our understanding of RNA methylation and its impact on gene regulation. Let's explore some real-world applications:

Transcriptome-wide Profiling:

RNA bisulfite sequencing enables the identification and quantification of m5C modifications across the entire transcriptome. This comprehensive profiling offers insights into the dynamics of RNA methylation, revealing patterns associated with different developmental stages, tissues, and disease conditions.

Functional Characterization:

Researchers can investigate the functional consequences of m5C modifications by correlating RNA methylation patterns with gene expression. Understanding how RNA methylation influences mRNA stability, alternative splicing, translation efficiency, and RNA-protein interactions provides valuable insights into post-transcriptional gene regulation.

Disease Mechanisms:

Dysregulation of RNA methylation has been implicated in various diseases, including cancer, neurological disorders, and metabolic disorders. Bisulfite sequencing on RNA helps identify specific m5C modifications associated with disease states, helping identify potential markers for diagnosis and targets for treatment.

How have we used bisulfite sequencing to study the role of METTL15 and NSUN2 in mitochondria? 

In 2019, we used bisulfite sequencing and other techniques to show that METTL15 is the main N4-methylcytidine (m4C) methyltransferase in human cells and that it is responsible for methylating position C839 in mitochondrial 12S rRNA. Without METTL15, the assembly of the mitoribosome decreases.

We also used bisulfite sequencing to study NSUN2 in mitochondria and show that generating m5C at several positions of mitochondrial tRNAs is necessary. You can see some examples of our RNA methylation analysis here.

Would you like help analysing your data? Or would you like advice on how to study m5C/m4C in your research projects? Contact us for more information about what we can offer!