RNA editing and autophagy in Drosophila melanogaster

Post-transcriptional regulation of gene expression involves a diverse set of mechanisms such as RNA splicing, RNA localization, and RNA turn-over. Adenosine to Inosine (A-to-I) RNA editing is an additional post-transcriptional regulatory mechanism. Temporally, it occurs after transcription and before RNA splicing and has been shown in some instances to possibly modulate alternative splicing events. This is the case for example, with the pre-mRNA encoding the GluR- 2 subunit of AMPA receptor, a glutamate-activated ion channel. ADAR (Adenosine deaminase acting on RNA) proteins bind to double-stranded regions in pre-messenger RNAs. They deaminate specific adenosines, generating inosines; if the editing event occurs within the coding region, inosine is then interpreted as guanosine by the ribosomal translational machinery, changing codon meaning. These editing events can increase the repertoire of translated proteins, generating molecular diversity and modifying protein function. In mammals there are four ADAR genes: ADAR1, ADAR2, ADAR3 and TENR. ADAR3 and TENR are enzymatically inactive. All the proteins have two types of functional domains: (i) the catalytic deaminase domain at the carboxyl-terminus and (ii) the double stranded RNA binding domains, dsRBDs, at the amino terminus. ADAR1 and ADAR2 differ significantly at the amino terminus, by the number of the dsRNA binding domains (three and two dsRBDs for ADAR1 and ADAR2 protein, respectively). The differences observed between ADAR1 and ADAR2 are likely to reflect the different repertoires of substrates edited by these two enzymes. Data concerning the conservation of Adar genes throughout evolution suggest that Drosophila melanogaster has a unique Adar gene which is a true ortholog of human ADAR2 rather than an invertebrate gene ancestral for both vertebrate genes. Flies that are null mutants for Adar (Adar5G1 mutants) display profound behavioral and locomotive deficits. Impairment in motor activity of the mutants is succeeded by age-dependent neurodegeneration, characterized by swelling within the Adar-null mutant fly brain. The initial focus of my thesis was to elucidate what causes Adar mutant phenotypes or, whether it is possible, to suppress them. I took advantage of Drosophila genetics to establish a forward genetic screen for suppressors of reduced Adar5G1 viability which is approximately 20-30% in comparison to control flies at eclosion. The results from an interaction screen on Chromosome 2L were further confirmed using Exelixis P-element insertion lines. The screen revealed that decreasing Tor (Target of rapamycin) expression suppresses Adar mutant phenotypes. TOR plays a role in maintaining cellular homeostasis by balancing the metabolic processes. It controls anabolic events by phosphorylating eukaryotic translation initiation factor 4E-binding protein (4E-BP) and p70 S6 kinase (S6K) and inducing cap-mediated translation. However, different types of stress, signals or increased demand in catabolic processes, converge to reduce TOR enzymatic activity. This results in long-lived proteins and organelles being engulfed in double-membrane vesicles and degraded; this bulk degradation process is called (macro)autophagy. The second aim of my thesis was to clarify which pathway, downstream to TOR, was responsible for the suppression of Adar-null phenotypes. I mimicked the effect of reduced Tor expression by manipulating genetically the cap-dependent translation and the autophagy pathways. Interestingly, boosting the expression of Atg (autophagy specific genes) genes, such as, Atg1 and Atg5, thereby increasing the activation rate of the autophagy pathway, suppresses Adar5G1 phenotypes. Finally, I found that Adar5G1 mutant flies have an increased level of autophagy that is observable from the larval stage. I investigated possible stresses affecting our mutants; Adar-mutant larval fat cells show ER stress triggering an unfolded protein response as indicated by expression of XbpI-eGFP reporter. Thus, ER stress might induce increased autophagy and it can lead to locomotive impairments and neurodegeneration in Adar-null mutants. These results suggest a function for the Adar gene in regulating cellular stress

Membrane and synaptic defects leading to neurodegeneration in Adar mutant Drosophila are rescued by increased autophagy

BackgroundIn fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration.ResultsA genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested.ConclusionsThese findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage

Functional conservation in human and Drosophila of Metazoan ADAR2 involved in RNA editing: loss of ADAR1 in insects

Flies with mutations in the single Drosophila Adar gene encoding an RNA editing enzyme involved in editing 4% of all transcripts have severe locomotion defects and develop age-dependent neurodegeneration. Vertebrates have two ADAR-editing enzymes that are catalytically active; ADAR1 and ADAR2. We show that human ADAR2 rescues Drosophila Adar mutant phenotypes. Neither the short nuclear ADAR1p110 isoform nor the longer interferon-inducible cytoplasmic ADAR1p150 isoform rescue walking defects efficiently, nor do they correctly edit specific sites in Drosophila transcripts. Surprisingly, human ADAR1p110 does suppress age-dependent neurodegeneration in Drosophila Adar mutants whereas ADAR1p150 does not. The single Drosophila Adar gene was previously assumed to represent an evolutionary ancestor of the multiple vertebrate ADARs. The strong functional similarity of human ADAR2 and Drosophila Adar suggests rather that these are true orthologs. By a combination of direct cloning and searching new invertebrate genome sequences we show that distinct ADAR1 and ADAR2 genes were present very early in the Metazoan lineage, both occurring before the split between the Bilateria and Cnidarians. The ADAR1 gene has been lost several times, including during the evolution of insects and crustacea. These data complement our rescue results, supporting the idea that ADAR1 and ADAR2 have evolved highly conserved, distinct functions

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects

Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors