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.

Pre-mRNA-splicing and adenosine to inosine (A-to-I) RNA-editing occur mostly cotranscriptionally. During A-to-I editing, a genomically encoded adenosine is deaminated to inosine by adenosine deaminases acting on RNA (ADARs). Editing-competent stems are frequently formed between exons and introns. Consistently, studies using reporter assays have shown that splicing efficiency can affect editing levels. Here, we use Nascent-seq and identify ∼90,000 novel A-to-I editing events in the mouse brain transcriptome. Most novel sites are located in intronic regions. Unlike previously assumed, we show that both ADAR (ADAR1) and ADARB1 (ADAR2) can edit repeat elements and regular transcripts to the same extent. We find that inhibition of splicing primarily increases editing levels at hundreds of sites, suggesting that reduced splicing efficiency extends the exposure of intronic and exonic sequences to ADAR enzymes. Lack of splicing factors NOVA1 or NOVA2 changes global editing levels, demonstrating that alternative splicing factors can modulate RNA editing. Finally, we show that intron retention rates correlate with editing levels across different brain tissues. We therefore demonstrate that splicing efficiency is a major factor controlling tissue-specific differences in editing levels.

Adenosine-to-inosine RNA editing and pre-mRNA splicing largely occur cotranscriptionally and influence each other. Here, we use mice deficient in either one of the two editing enzymes ADAR (ADAR1) or ADARB1 (ADAR2) to determine the transcriptome-wide impact of RNA editing on splicing across different tissues. We find that ADAR has a 100× higher impact on splicing than ADARB1, although both enzymes target a similar number of substrates with a large common overlap. Consistently, differentially spliced regions frequently harbor ADAR editing sites. Moreover, catalytically dead ADAR also impacts splicing, demonstrating that RNA binding of ADAR affects splicing. In contrast, ADARB1 editing sites are found enriched 5′ of differentially spliced regions. Several of these ADARB1-mediated editing events change splice consensus sequences, therefore strongly influencing splicing of some mRNAs. A significant overlap between differentially edited and differentially spliced sites suggests evolutionary selection toward splicing being regulated by editing in a tissue-specific manner.

Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon–intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing.

The RNA-editing protein ADAR is essential for early development in the mouse. Genetic evidence suggests that A to I editing marks endogenous RNAs as ‘self’. Today, different Adar knockout alleles have been generated that show a common phenotype of apoptosis, liver disintegration, elevated immune response and lethality at E12.5. All the Adar knockout alleles can be rescued by a concomitant deletion of the innate immunity genes Mavs or Ifih1 (MDA5), albeit to different extents. This suggests multiple functions of ADAR. We analyze Adar^Δ7-9 mice that show a unique growth defect phenotype when rescued by Mavs. We show that Adar^Δ7-9 can form a truncated, unstable, editing deficient protein that is mislocalized. Histological and hematologic analysis of these mice indicate multiple tissue- and hematopoietic defects. Gene expression profiling shows dysregulation of Rps3a1 and Rps3a3 in rescued Adar^Δ7-9. Consistently, a distortion in 40S and 60S ribosome ratios is observed in liver cells. This dysregulation is also seen in Adar^Δ2-13; Mavs^−/− but not in Adar^E861A/E861A; Ifih1^−/− mice, suggesting editing-independent functions of ADAR in regulating expression levels of Rps3a1 and Rps3a3. In conclusion, our study demonstrates the importance of ADAR in post-natal development which cannot be compensated by ADARB1.