Understanding RNA modifications: the promises and technological bottlenecks of the 'epitranscriptome'

Abstract

The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the 'epitranscriptome'. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications.

Keywords: RNA; chemical modification; epigenetics; transcriptome.

Figure 1.

Modifications under the surface of detection. Improved sequencing methods have led to the discovery of millions of modification sites in all classes of RNAs. However, efficient detection of modifications is mostly possible at sites that undergo deamination. Given that more than 130 types of modified ribonucleotides are known to date, it can be expected that novel technologies will lead to a huge increase in detectable RNA modifications that are currently hidden underneath the (detection) surface.

Figure 2.

Advances in conceptualizing the dynamic ‘epitranscriptome’. While sequence context-dependent mapping has become a technical reality (a), it is currently not clear how RNA modifications influence each other. Some (but not all) modifications have been shown to be reversible (b), while other modifications have been shown to be inducible (c). The mechanisms of induction and removal of modifications, and in particular the regulatory mechanisms underlying the dynamic landscape of RNA modifications, are poorly understood. As some RNA modifications are specific for certain phyla, their presence or absence can be interpreted as pathogen-associated molecular patterns (PAMPs) and therefore help to distinguish self- from non-self RNAs (d).

Figure 3.

The challenges of the multi-layered world of RNA modifications. RNA modifications are introduced by ‘writer’ enzymes that can then be interpreted by ‘reader’ proteins before being physically removed by ‘erasers’ or further modified by ‘modifiers’ of modifications (a). While few modifications have been described that employ all three classes of proteins, it is conceivable that readers and erasers exist for many more modification types than is currently appreciated. Moreover, all classes of proteins may exhibit moonlighting functions (b), in addition to their reported modification-related activity. The majority of RNA modifications occur in abundant RNA species, such as rRNAs (greater than 90% of all RNAs) and tRNAs (greater than 5% of all RNAs), but also non-abundant RNAs (less than 5% of all RNAs including cRNA, small RNAs, lncRNAs, circRNAs) carry modifications (c). Presently, it is impossible to distinguish whether a given RNA modification in a population of cells is very abundant in some but completely absent from other cells, or alternatively, whether all cells in a population contain a rather low abundance of this particular modification (d). Lastly, even within individual cells, it is unclear whether individual RNAs carry multiple marks of the same modification, while other RNAs remain unmodified, or alternatively, whether modifications are distributed rather homogeneously among all transcripts (e). Addressing these questions will be the next challenge in RNA modification research.