Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila.

The accurate and thorough genome-wide detection of adenosine-to-inosine editing, a biologically indispensable process, has proven challenging. Here, we present a discovery pipeline in adult Drosophila, with 3,581 high-confidence editing sites identified with an estimated accuracy of 87%. The target genes and specific sites highlight global biological properties and functions of RNA editing, including hitherto-unknown editing in well-characterized classes of noncoding RNAs and 645 sites that cause amino acid substitutions, usually at conserved positions. The spectrum of functions that these gene targets encompass suggests that editing participates in a diverse set of cellular processes. Editing sites in Drosophila exhibit sequence-motif preferences and tend to be concentrated within a small subset of total RNAs. Finally, editing regulates expression levels of target mRNAs and strongly correlates with alternative splicing.

RNA editing in eag potassium channels: biophysical consequences of editing a conserved S6 residue.

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2-4, within the cytoplasmic C-terminal domain). We mutated these residues individually and expressed the channels in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg(+2) concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg(+2) than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel's function.

Integration of a retrograde signal during synapse formation by glia-secreted TGF-ß ligand.

Glial cells are crucial regulators of synapse formation, elimination, and plasticity [1, 2]. In vitro studies have begun to identify glial-derived synaptogenic factors [1], but neuron-glia signaling events during synapse formation in vivo remain poorly defined. The coordinated development of pre- and postsynaptic compartments at the Drosophila neuromuscular junction (NMJ) depends on a muscle-secreted retrograde signal, the TGF-ß/BMP Glass bottom boat (Gbb) [3, 4]. Muscle-derived Gbb activates the TGF-ß receptors Wishful thinking (Wit) and either Saxophone (Sax) or Thick veins (Tkv) in motor neurons [3, 4]. This induces phosphorylation of Mad (P-Mad) in motor neurons, its translocation into the nucleus with a co-Smad, and activation of transcriptional programs controlling presynaptic bouton growth [5]. Here we show that NMJ glia release the TGF-ß ligand Maverick (Mav), which likely activates the muscle activin-type receptor Punt to potently modulate Gbb-dependent retrograde signaling and synaptic growth. Loss of glial Mav results in strikingly reduced P-Mad at NMJs, decreased Gbb transcription in muscle, and in turn reduced muscle-to-motor neuron retrograde TGF-ß/BMP signaling. We propose that by controlling Gbb release from muscle, glial cells fine tune the ability of motor neurons to extend new synaptic boutons in correlation to muscle growth. Our work identifies a novel glia-derived synaptogenic factor by which glia modulate synapse formation in vivo.

Regulated RNA editing and functional epistasis in Shaker potassium channels.

Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 2(4) = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

Characterization of five RNA editing sites in Shab potassium channels.

RNA editing revises the genetic code at precise locations, creating single base changes in mRNA. These changes can result in altered coding potential and modifications to protein function. Sequence analysis of the Shab potassium channel of Drosophila melanogaster revealed five such RNA editing sites. Four are constitutively edited (I583V, T643A, Y660C and I681V) and one undergoes developmentally regulated editing (T671A). These sites are located in the S4, S5-S6 loop and the S6 segments of the channel. We examined the biophysical consequences of editing at these sites by creating point mutations, each containing the genomic (unedited) base at one of the five sites in the background of a channel in which all other sites are edited. We also created a completely unedited construct. The function of these constructs was characterized using two-microelectrode voltage clamp in Xenopus oocytes. Each individual 'unediting' mutation slowed the time course of deactivation and the rise time during channel activation. Two of the mutants exhibited significant hyperpolarized shifts in their midpoints of activation. Constructs that deactivated slowly also inactivated slowly, supporting a mechanism of closed-state inactivation. One of the editing sites, position 660, aligns with the Shaker 449 residue, which is known to be important in tetraethylammonium (TEA) block. The aromatic, genomically-encoded residue tyrosine at this position in Shab enhances TEA block 14 fold compared to the edited residue, cysteine. These results show that both the position of the RNA editing site and the identity of the substituted amino acid are important for channel function.