RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine ...into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.
Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification that affects the information encoded from DNA to RNA to protein. RNA editing can generate a multitude of ...transcript isoforms and can potentially be used to optimize protein function in response to varying conditions. In light of this and the fact that millions of editing sites have been identified in many different species, it is interesting to examine the extent to which these sites have evolved to be functionally important. In this review, we discuss results pertaining to the evolution of RNA editing, specifically in humans, cephalopods, and Drosophila. We focus on how comparative genomics approaches have aided in the identification of sites that are likely to be advantageous. The use of RNA editing as a mechanism to adapt to varying environmental conditions will also be reviewed.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Adenosine-to-inosine RNA editing diversifies the transcriptome and promotes functional diversity, particularly in the brain. A plethora of editing sites has been recently identified; however, how ...they are selected and regulated and which are functionally important are largely unknown. Here we show the cis-regulation and stepwise selection of RNA editing during Drosophila evolution and pinpoint a large number of functional editing sites. We found that the establishment of editing and variation in editing levels across Drosophila species are largely explained and predicted by cis-regulatory elements. Furthermore, editing events that arose early in the species tree tend to be more highly edited in clusters and enriched in slowly-evolved neuronal genes, thus suggesting that the main role of RNA editing is for fine-tuning neurological functions. While nonsynonymous editing events have been long recognized as playing a functional role, in addition to nonsynonymous editing sites, a large fraction of 3'UTR editing sites is evolutionarily constrained, highly edited, and thus likely functional. We find that these 3'UTR editing events can alter mRNA stability and affect miRNA binding and thus highlight the functional roles of noncoding RNA editing. Our work, through evolutionary analyses of RNA editing in Drosophila, uncovers novel insights of RNA editing regulation as well as its functions in both coding and non-coding regions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and ...efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
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•sgRNAs have limited off-target effects at loci with three or more mismatches•Efficiency correlates with the sgRNA GC content 6 nt adjacent to PAMs•Enhanced efficiency allows for the mutation of four genes together at high rates•Homology-directed repair rates are significantly higher than previously reported
Ren et al. examine sgRNA properties that enhance efficiency of CRISPR/Cas9 in Drosophila. They demonstrate that such optimization allows more efficient mutagenesis of multiple genes and of homozygous lethal genes through homology-directed repair.
ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila Adar
...mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
Adenosine-to-inosine RNA editing modifies maturing mRNAs through the binding of adenosine deaminase acting on RNA (Adar) proteins to double-stranded RNA structures in a process critical for neuronal ...function. Editing levels at individual editing sites span a broad range and are mediated by both cis-acting elements (surrounding RNA sequence and secondary structure) and trans-acting factors. Here, we aim to determine the roles that cis-acting elements and trans-acting factors play in regulating editing levels. Using two closely related Drosophila species, D. melanogaster and D. sechellia, and their F1 hybrids, we dissect the effects of cis sequences from trans regulators on editing levels by comparing species-specific editing in parents and their hybrids. We report that cis sequence differences are largely responsible for editing level differences between these two Drosophila species. This study presents evidence for cis sequence and structure changes as the dominant evolutionary force that modulates RNA editing levels between these Drosophila species.
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•RNA editing levels differ between D. melanogaster and D. sechellia•Most editing level differences are maintained in species-specific F1 hybrid alleles•Cis sequences surrounding editing sites largely affect editing levels•Higher editing levels correlate with more stable dsRNA structures
Sapiro et al. measure RNA editing levels at hundreds of editing sites in two Drosophila species and their F1 hybrids to dissect cis versus trans regulation of editing levels. They show that many editing level differences between the species are due to cis sequence and structure changes of edited pre-mRNAs.
We show that RNA editing sites can be called with high confidence using RNA sequencing data from multiple samples across either individuals or species, without the need for matched genomic DNA ...sequence. We identified many previously unidentified editing sites in both humans and Drosophila; our results nearly double the known number of human protein recoding events. We also found that human genes harboring conserved editing sites within Alu repeats are enriched for neuronal functions.
Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular ...cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs in vitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.
Adenosine-to-inosine (A-to-I) RNA editing, catalysed by ADAR enzymes conserved in metazoans, plays an important role in neurological functions. Although the fine-tuning mechanism provided by A-to-I ...RNA editing is important, the underlying rules governing ADAR substrate recognition are not well understood. We apply a quantitative trait loci (QTL) mapping approach to identify genetic variants associated with variability in RNA editing. With very accurate measurement of RNA editing levels at 789 sites in 131 Drosophila melanogaster strains, here we identify 545 editing QTLs (edQTLs) associated with differences in RNA editing. We demonstrate that many edQTLs can act through changes in the local secondary structure for edited dsRNAs. Furthermore, we find that edQTLs located outside of the edited dsRNA duplex are enriched in secondary structure, suggesting that distal dsRNA structure beyond the editing site duplex affects RNA editing efficiency. Our work will facilitate the understanding of the cis-regulatory code of RNA editing.
Many important modifications are used to tweak genomically-encoded information as it is being transcribed and translated. One type of RNA modification, adenosine-to-inosine (A-to-I) RNA editing, is a ...process in which the adenosine deaminase acting on RNA (ADAR) proteins catalyze the conversion of certain adenosines to inosines, which are treated by the cellular machinery as guanosines. A-to-I RNA editing is essential for the neurological and immune well-being of many animals, and it is a widespread process: There are thousands to millions of editing sites across the transcriptomes of flies and humans, respectively. However, it is largely unknown which sites are important, what their individual functions are, and what makes particular adenosines edited. In this dissertation, Drosophila is used as a model organism to explore key questions about the function and evolution of RNA editing, and the characteristics of RNA editing substrates. First, in Chapter 2, I describe the generation and characterization of an ADAR deaminase inactive point mutant fly and find that Drosophila ADAR has both neurological and immune RNA editing-dependent functions. Secondly, in Chapters 3 and 4, I explore the characteristics of edited RNA. I describe a computational pipeline used to predict the regulatory sequences base-paired to regions that undergo RNA editing and derive rules about RNA editing substrates, through an examination of the changes in RNA sequence and structure associated with changes in RNA editing across lines of Drosophila melanogaster (Chapter 3), and across Drosophila species (Chapter 4). Furthermore, a large fraction of nonsynonymous and noncoding editing events are evolutionarily constrained and therefore likely functionally important. Finally, in Chapter 5, I explore the functions of individual RNA editing events in vivo, through the generation and characterization of RNA editing event-specific mutants. These mutants validated some RNA structure predictions from Chapter 3. Furthermore, they do not have the severe phenotypes of the Adar mutants described in Chapter 2, suggesting that the editing events perturbed here could have subtle or redundant functions. Overall, this dissertation provides insights into the function and evolution of RNA editing events and their substrates.
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