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.

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.

Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.

Methyltranscriptome is an exciting new area that studies the mechanisms and functions of methylation in transcripts. A knowledge base with the systematic collection and curation of context specific transcriptome-wide methylations is critical for elucidating their biological functions as well as for developing bioinformatics tools. Since its inception in 2014, the Met-DB (Liu, H., Flores, M.A., Meng, J., Zhang, L., Zhao, X., Rao, M.K., Chen, Y. and Huang, Y. (2015) MeT-DB: a database of transcriptome methylation in mammalian cells. Nucleic Acids Res., 43, D197-D203), has become an important resource for methyltranscriptome, especially in the N6-methyl-adenosine (m6A) research community. Here, we report Met-DB v2.0, the significantly improved second version of Met-DB, which is entirely redesigned to focus more on elucidating context-specific m6A functions. Met-DB v2.0 has a major increase in context-specific m6A peaks and single-base sites predicted from 185 samples for 7 species from 26 independent studies. Moreover, it is also integrated with a new database for targets of m6A readers, erasers and writers and expanded with more collections of functional data. The redesigned Met-DB v2.0 web interface and genome browser provide more friendly, powerful, and informative ways to query and visualize the data. More importantly, MeT-DB v2.0 offers for the first time a series of tools specifically designed for understanding m6A functions. Met-DB V2.0 will be a valuable resource for m6A methyltranscriptome research. The Met-DB V2.0 database is available at http://compgenomics.utsa.edu/MeTDB/ and http://www.xjtlu.edu.cn/metdb2.

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids.

The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of ADARs. Here, we provide evidence for a molecular mechanism that might control nuclear import of specific ADARs and, in turn, nuclear RNA editing. We identify an in vivo ADAR3 interaction partner, importin alpha 1 (KPNA2) that specifically recognizes an arginine-rich ADAR3 sequence motif and show that it acts as a functional nuclear localization sequence. Furthermore, whereas KPNA2, but not KPNA1 or KNPA3, recognizes the ADAR3 NLS, we observe the converse binding specificity with ADAR2. Interestingly, alternative splicing of ADAR2 pre-mRNA introduces an ADAR3-like NLS that alters the interaction profile with the importins. Thus, in vivo RNA editing might be regulated, in part, through controlled subcellular localization of ADARs, which in turn is governed by the coordinated local expression of importin alpha proteins and ADAR protein variants.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.

Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3'-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

In all organisms, transfer RNAs (tRNAs) undergo extensive post-transcriptional modifications. Although base modifications in the anticodon are known to alter decoding specificity or improve decoding accuracy, much less is known about the functional relevance of modifications in other positions of tRNAs. Here, we report the identification of an A-to-I tRNA editing enzyme that modifies the tRNA-Ala(AGC) in the model plant Arabidopsis thaliana. The enzyme is homologous to Tad1p, a yeast tRNA-specific adenosine deaminase, and it selectively deaminates the adenosine in the position 3'-adjacent to the anticodon (A37) to inosine. We show that the AtTAD1 protein is exclusively localized in the nucleus. The tad1 loss-of-function mutants isolated in Arabidopsis show normal accumulation of the tRNA-Ala(AGC), suggesting that the loss of the I37 modification does not affect tRNA stability. The tad1 knockout mutants display no discernible phenotype under standard growth conditions, but produce less biomass under environmental stress conditions. Our results provide the first evidence in support of a physiological relevance of the A37-to-I modification in eukaryotes.

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors.

Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.

β-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Adenosine deaminases-acting-on-RNA (ADAR) proteins induce adenosine-to-inosine editing in double-stranded RNA molecules. This editing generates RNA diversity at the post-transcriptional level, and it has been implicated in the control of cell differentiation and development. The editing of microRNA (miRNA) precursors, along with Tudor-SN (Snd1) activity, could lead to the elimination of selected miRNAs and reprogram miRNA activity. Here, we report the dynamics of adenosine-to-inosine editing in miRNA precursors and their selected elimination during mouse preimplantation development. Adar1p110 and Snd1 were found to be strongly but differentially expressed in oocytes and zygotes with respect to later pre-implantation stages. When the biogenesis of miR-151 was assessed, the majority of miR-151 precursors was edited and subsequently eliminated during early development. Deep sequencing of this and other miRNAs confirmed that, in general, edited precursors were selectively eliminated at early post-zygotic stages. Moreover, in oocytes and throughout the zygote-to-blastocyst stages, Tudor-SN accumulated in newly discovered aggregates termed 'T bodies'. These results provide new insight into how editing and Tudor-SN-mediated elimination of miRNA precursors is regulated during early development.

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N8-(2'deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.