Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus.

BACKGROUND: Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. RESULTS: Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. CONCLUSIONS: Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal's unique physiology or environmental adaptation.

Identification of Bona Fide RNA Editing Sites: History, Challenges, and Opportunities.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of enzymes of which there are three members (ADAR1, ADAR2, and ADAR3), is a major gene regulatory mechanism that diversifies the transcriptome. It is widespread in many metazoans, including humans. As inosine is interpreted by cellular machineries mainly as guanosine, A-to-I editing effectively gives A-to-G nucleotide changes. Depending on its location, an editing event can generate new protein isoforms or influence other RNA processing pathways. Researchers have found that ADAR-mediated editing performs diverse functions. For example, it enables living organisms such as cephalopods to adapt rapidly to fluctuating environmental conditions such as water temperature. In development, the loss of ADAR1 is embryonically lethal partly because endogenous double-stranded RNAs (dsRNAs) are no longer marked by inosines, which signal "self", and thus cause the melanoma differentiation-associated protein 5 (MDA5) sensor to trigger a deleterious interferon response. Hence, ADAR1 plays a key role in preventing aberrant activation of the innate immune system. Furthermore, ADAR enzymes have been implicated in myriad human diseases. Intriguingly, some cancer cells are known to exploit ADAR1 activity to dodge immune responses. However, the exact identities of immunogenic RNAs in different biological contexts have remained elusive. Consequently, there is tremendous interest in identifying inosine-containing RNAs in the cell.The identification of A-to-I RNA editing sites is dependent on the sequencing of nucleic acids. Technological and algorithmic advancements over the past decades have revolutionized the way editing events are detected. At the beginning, the discovery of editing sites relies on Sanger sequencing, a first-generation technology. Both RNA, which is reverse transcribed into complementary DNA (cDNA), and genomic DNA (gDNA) from the same source are analyzed. After sequence alignment, one would require an adenosine to be present in the genome but a guanosine to be detected in the RNA sample for a position to be declared as an editing site. However, an issue with Sanger sequencing is its low throughput. Subsequently, Illumina sequencing, a second-generation technology, was invented. By permitting the simultaneous interrogation of millions of molecules, it enables many editing sites to be identified rapidly. However, a key challenge is that the Illumina platform produces short sequencing reads that can be difficult to map accurately. To tackle the challenge, we and others developed computational workflows with a series of filters to discard sites that are likely to be false positives. When Illumina sequencing data sets are properly analyzed, A-to-G variants should emerge as the most dominant mismatch type. Moreover, the quantitative nature of the data allows us to build a comprehensive atlas of editing-level measurements across different biological contexts, providing deep insights into the spatiotemporal dynamics of RNA editing. However, difficulties remain in identifying true A-to-I editing sites in short protein-coding exons or in organisms and diseases where DNA mutations and genomic polymorphisms are prevalent and mostly unknown. Nanopore sequencing, a third-generation technology, promises to address the difficulties, as it allows native RNAs to be sequenced without conversion to cDNA, preserving base modifications that can be directly detected through machine learning. We recently demonstrated that nanopore sequencing could be used to identify A-to-I editing sites in native RNA directly. Although further work is needed to enhance the detection accuracy in single molecules from fewer cells, the nanopore technology holds the potential to revolutionize epitranscriptomic studies.

Direct identification of A-to-I editing sites with nanopore native RNA sequencing.

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.

RNA Editing in Human and Mouse Tissues.

Adenosine-to-inosine (A-to-I) RNA editing is a fundamental posttranscriptional mechanism that greatly diversifies the transcriptome in many living organisms, including mammals. Multiple studies have demonstrated the importance of this process not just in normal development and physiology but also in various human diseases. Importantly, the precise editing level of a site may have downstream consequences on cellular behavior. Hence, the editing levels should be quantified as accurately as possible. In this chapter, we describe how to examine RNA editing in human and mouse tissues. The rapid development of next-generation sequencing technologies is affording us an unprecedented ability to accurately measure the editing levels of numerous sites simultaneously. Our experimental workflow includes the harvesting of high-quality RNA samples and the construction of different high-throughput sequencing libraries. We also delineate the computational steps needed to analyze the sequencing data from an Illumina platform.

A human expression system based on HEK293 for the stable production of recombinant erythropoietin.

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

SRSF9 selectively represses ADAR2-mediated editing of brain-specific sites in primates.

Adenosine-to-inosine (A-to-I) RNA editing displays diverse spatial patterns across different tissues. However, the human genome encodes only two catalytically active editing enzymes (ADAR1 and ADAR2), suggesting that other regulatory factors help shape the editing landscape. Here, we show that the splicing factor SRSF9 selectively controls the editing of many brain-specific sites in primates. SRSF9 is more lowly expressed in the brain than in non-brain tissues. Gene perturbation experiments and minigene analysis of candidate sites demonstrated that SRSF9 could robustly repress A-to-I editing by ADAR2. We found that SRSF9 biochemically interacted with ADAR2 in the nucleus via its RRM2 domain. This interaction required the presence of the RNA substrate and disrupted the formation of ADAR2 dimers. Transcriptome-wide location analysis and RNA sequencing revealed 1328 editing sites that are controlled directly by SRSF9. This regulon is significantly enriched for brain-specific sites. We further uncovered a novel motif in the ADAR2-dependent SRSF9 binding sites and provided evidence that the splicing factor prevents loss of cell viability by inhibiting ADAR2-mediated editing of genes involved in proteostasis, energy metabolism, the cell cycle and DNA repair. Collectively, our results highlight the importance of SRSF9 as an editing regulator and suggest potential roles for other splicing factors.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing.

CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.

Accurate identification of human Alu and non-Alu RNA editing sites.

We developed a computational framework to robustly identify RNA editing sites using transcriptome and genome deep-sequencing data from the same individual. As compared with previous methods, our approach identified a large number of Alu and non-Alu RNA editing sites with high specificity. We also found that editing of non-Alu sites appears to be dependent on nearby edited Alu sites, possibly through the locally formed double-stranded RNA structure.