RNA molecules are essential for carrying and using genetic information, but their function depends on more than just the sequence of their nucleotides. Chemical structures attached to RNA can influence how RNA is processed, protected, recognized, and broken down inside cells.

One important feature is the RNA cap, a chemical structure attached to the 5′ end of an RNA molecule. For many years, researchers believed that most eukaryotic messenger RNA was capped primarily with a molecule called 7-methylguanosine, or m7G. More recently, scientists have discovered several alternative, or noncanonical, RNA caps, suggesting that RNA molecules can carry a wider variety of chemical modifications than previously recognized.

Researchers at the Czech Academy of Sciences in Prague and colleagues have now identified two additional RNA cap structures in mammalian cells, diadenosine diphosphate, known as Ap2A, and adenosine guanosine diphosphate, known as Ap2G. Together, these structures are referred to as Ap2N caps.

Detecting a new type of RNA cap

To determine whether Ap2N caps are present on mammalian RNA, the researchers developed a method using liquid chromatography and mass spectrometry, or LC-MS.

Liquid chromatography separates molecules in a sample based on their chemical properties. Mass spectrometry then measures those molecules based on their mass and other characteristics. Together, these techniques allowed the researchers to detect and quantify RNA molecules carrying Ap2N caps.

The researchers also found that levels of Ap2N-capped RNA increased when cells were exposed to oxidative stress. Oxidative stress occurs when reactive molecules accumulate faster than cells can neutralize them, potentially damaging proteins, DNA, RNA, and other cellular components.

The increase in Ap2N-capped RNA under these conditions suggests that these unusual caps may be connected to how cells respond to environmental or physiological stress, although their precise biological role remains to be determined.

Developing a new RNA sequencing method

Detecting an RNA cap chemically is only part of the challenge. Researchers also need to determine which RNA molecules carry the cap.

To do this, the team developed a specialized RNA sequencing method called Ap2N-RNA Seq.

Ap2N-RNA Seq protocol

(A) Overview of the Ap2N-RNA Seq method. First, the isolated short RNA is either left untreated (Ap2N sample) or treated with NUDT12 to remove the Ap2N cap (Negative CTRL). Subsequently, all m7G capped RNAs are removed by incubation with the mRNA decapping enzyme (MDE) and 5′ exonuclease. Afterward, a short polyA tail is added by E. coli polyA polymerase. During this step, 70% of 3′ dATP (cordycepin) is added to the reaction to shield the 3′ end of RNA and avoid ligation and circularization in the subsequent step. Finally, an RNA adaptor is ligated to Ap2N-RNA by truncated T4 RNA ligase 2 K227Q, the resulting RNA is reverse transcribed using polyT oligo, amplified, and subjected to dsDNA Illumina sequencing. (B) RT-qPCR analysis of the products of the Ap2N-RNA Seq method applied to a model Ap2A-U1 snRNA. The protocol significantly enriches the Ap2A-U1 RNA compared to the negative control (Ap2A-U1 + NUDT12) or U1 RNAs capped with other caps (m7Gp3A and pppA). The significance values represent the adjusted P-value (ANOVA). (C) RNA-Seq results of Ap2N-RNA Seq applied to model Ap2A-U1 RNA. After removing the sequence of the 5′ adaptor and mapping the reads to the in vitro synthetized U1 reference, the 5′ end of the ligated RNA correctly maps to the +1 position of the U1 sequence in the Ap2N sample (green area) compared to ~50% less reads of the negative control sample (+NUDT12, brown area) with rather nonspecific distribution along the sequence.

The technique takes advantage of an enzyme called truncated T4 RNA Ligase 2. The enzyme can selectively attach an RNA adaptor to RNA molecules carrying Ap2N caps. Adaptors are short pieces of RNA that allow selected molecules to be prepared for sequencing.

By enriching for Ap2N-capped molecules before sequencing, researchers can identify the RNA sequences associated with these unusual caps.

When the researchers applied Ap2N-RNA Seq to short RNA molecules from human cells, they identified several RNA species derived from transfer RNA, or tRNA, as potential Ap2N-capped RNAs.

A closer look at transfer RNA fragments

Transfer RNAs are best known for their role in protein production. During translation, tRNAs carry amino acids to the ribosome, where proteins are assembled according to instructions encoded in messenger RNA.

Researchers now know that tRNAs can also be cut into smaller fragments. These tRNA-derived fragments are increasingly recognized as biologically active molecules rather than simply products of RNA degradation.

Using Ap2N-RNA Seq, Vopalensky and colleagues detected previously uncharacterized fragments originating from the 3′ ends of tRNAs. Their results were consistent with these fragments carrying Ap2A caps at their 5′ ends.

This observation adds another layer to scientists’ understanding of how tRNAs can be processed and chemically modified inside cells. It also raises new questions about whether Ap2N capping affects the stability, localization, or biological activity of tRNA-derived fragments.

Expanding the view of the RNA life cycle

RNA molecules undergo many changes from the time they are produced until they are eventually degraded. They can be cut, chemically modified, transported within the cell, translated into proteins, or converted into smaller regulatory RNAs.

The discovery of Ap2A and Ap2G caps on mammalian RNA expands the growing collection of chemical modifications that may influence this RNA life cycle.

Just as importantly, Ap2N-RNA Seq provides researchers with a method for identifying which RNA molecules carry these caps. Combining RNA sequencing with chemical methods such as LC-MS could help researchers investigate when these caps appear, how their levels change under different cellular conditions, and what functions they may serve.

The findings add to growing evidence that RNA chemistry is considerably more diverse than the traditional view of RNA molecules carrying a standard set of modifications.