Decoding the Mysteries of RNA Methylation: Exploring Bisulfite Sequencing

In the fascinating world of genetics, understanding the mechanisms that regulate gene expression is crucial. The addition of chemical modifications to RNA molecules, known as RNA methylation. Bisulfite sequencing, originally 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 the 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 studies modifications are N6-methyladenosine (m6A), pseudouridine and 5-methylcytidine (m5C). The latter describes the addition of a methylgroup to the cytosine C5 atom.

Methylation Patterns and Gene Regulation

Methylation patterns have a profound influence on gene expression. Hypermethylation, which involves the addition of methyl groups, often occurs in regions known as CpG islands located near gene promoters. This dense methylation can inhibit transcription factors from binding to DNA, resulting in gene silencing. In contrast, hypomethylation, or the removal of methyl groups, is associated with increased gene expression. By mapping and understanding these patterns, researchers can gain insights into the regulation of specific genes and their involvement in various diseases. 

Understanding Bisulfite Sequencing

Bisulfite sequencing is a technique used to analyze the m5C (and to a lesser extend m4C) methylation status of RNA molecules.

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 revolutionized 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:

By correlating RNA methylation patterns with gene expression, researchers can investigate the functional consequences of m5C modifications. 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 methylation of position C839 in mitochondrial 12S rRNA. Without METTL15, the assembly of the mitoribosome is decreased.

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

Do you need help analysing your data? Or do you need advice on how to study m5C/m4C in your research projects? Just contact us for more information about what we can offer!