InteractiveFly: GeneBrief

no-on-and-no-off transient C: Biological Overview | References

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function (Long, 2010).

Post-transcriptional regulation of gene expression has a crucial role in the development, maintenance and plasticity of neuronal synapses. At the mRNA level, translation control in soma as well as remotely in dendrites and axonal growth cones regulates pathfinding, synaptogenesis and synaptic function. Neuronal mRNA transport and translation is often mediated by interaction between the 3' untranslated region (3'UTR) 'zipcode' and mRNA-binding proteins such as zip-code-binding protein (ZBP) and cytoplasmic polyadenylation-element-binding protein (CPEB). Other mRNA-binding proteins, such as fragile-X mental retardation protein (FMRP), similarly have crucial roles in the regulation of synaptic mRNA stability, trafficking and translation. Moreover, local protein degradation via the ubiquitin proteasome system (UPS) has also recently been established as a key mechanism that shapes synaptic structural development, neurotransmission strength and synaptic plasticity (Long, 2010 and references therein).

The Caenorhabditis elegans and Drosophila genetic systems have provided vital insights into the mechanisms of post-transcriptional regulation in sculpting synaptic properties. Drosophila FMRP is involved in mRNA trafficking and stability, and the activity-dependent regulation of mRNA translation. At the synapse, FMRP has several functions, including the control of axonal and dendritic arbor size, bouton number and distribution, transmission strength, postsynaptic glutamate receptor trafficking and the regulation of presynaptic vesicle pools. In balance with translation regulation, UPS-mediated degradation has dynamic functions that control synapse architecture, neurotransmission strength and synaptic protein abundance, including postsynaptic glutamate receptors. The ubiquitin ligase highwire acts to restrict synaptic overgrowth by down-regulating the MAPKKK–Wallenda pathway, where mutants exhibit increased neuromuscular junction (NMJ) branch and bouton numbers. Loss of C. elegans rpm-1 similarly causes disruption of synapse architecture and function. RPM-1 negatively regulates the MAP kinase pathway of MAPKKK DLK-1, MAPKK MKK-4 and p38 MAPK PMK-3. Recently, this pathway was shown to also regulate trafficking of the AMPA-type glutamate receptor GLR-1. Destabilization of enhancer binding protein CEBP-1 mRNA has also recently been shown to alter local translation of MAPKKK components in distal axons to disrupt axon morphology and synapse formation in C. elegans. Other recent work has shown the importance of microRNA pathways and processing bodies (P-bodies) (Long, 2010 and references therein).

Nonsense-mediated decay (NMD), an mRNA surveillance system that ensures message integrity by degrading transcripts containing nonsense mutations (Bramham, 2008; Giorgi, 2007), represents a relatively unexplored mechanism for regulating mRNA stability in neurons. Importantly, NMD is also proposed to regulate normal transcript expression, and so provide an alternative mechanism of control. Seven NMD pathway 'suppressor with morphogenetic effect on genitalia' (Smg) genes were originally identified in C. elegans (Hodgkin, 1989), with six gene homologs subsequently identified in Drosophila (Gatfield, 2004; Metzstein, 2006). It was recently demonstrated that eIF4AIII, a core exon junction complex (EJC) component, is associated with neuronal mRNA granules (Barbee, 2006; Giorgi, 2007; Shibuya, 2004; Shibuya, 2006). In mammals, the EJC is thought to have a crucial role in directing transcripts to the NMD pathway. This system controls glutamate receptor expression and long-term potentiation at the synapse via regulation of Arc protein expression (Bramham, 2008; Giorgi, 2007; Waung, 2008). Predicted NMD pathway targets include a number of synaptic genes; however, the functional requirement of NMD in maintaining the synapse has not before been directly tested in vivo (Long, 2010).

Classical Drosophila forward genetic screens to generate mutants with defective adult eye electroretinograms resulted in isolation of a pool of mutants with profoundly disrupted photoreceptor synaptic transmission. These genes were targeted to identify novel mechanisms required for synapse formation and function. Cloning and genomic rescue of the no-on-and-off transient C (nonC) mutant from this pool showed it to be the phosphatidylinositol 3-kinase-like kinase (PIKK) Smg1 gene, the regulative kinase of the NMD pathway. Detailed analyses of the NMJ synapse and a range of Smg1 alleles showed defects in morphological architecture and impaired synaptic function owing to disrupted synaptic vesicle cycling. Similarly, mutation of other key NMD pathway components, including Upf2 (Smg3) and Smg6 mutants that abrogate NMD and cause accumulation of PTC-containing transcripts (Metzstein, 2006), comparably impairs presynaptic function. These results demonstrate that NMD mRNA regulation is crucial for the proper development of synaptic architecture and for the maintenance of synaptic transmission efficacy (Long, 2010).

The ERG consists of a corneal-negative (downward) sustained component of summed photoreceptor phototransduction, and two transient components at lights-on and lights-off arising from neurotransmission in the lamina. The on-transient, in particular, corresponds directly to responses of laminar neurons to synaptic input from photoreceptors R1-R6. In wild-type animals, the peak amplitude of the sustained component increased with the strength of the light stimulus in a semi-log-dependent manner over a 3 log unit range of stimulus intensity. Synaptic transient amplitudes were lower only at the weakest light stimulus intensity, and then increased to a constant level over an increase in light intensity of three orders of magnitude. The peak amplitude of the phototransduction component and the synaptic on- and off-transient amplitudes were quantified and plotted as a function of the stimulus intensity (Long, 2010).

In the nonC mutant isolated in the genetic screen for photoreception mutants (nonC^P37), robust phototransduction persisted, but the synaptic transients were not detectable. Neither on-transients nor off-transients of any significant amplitude could be elicited from the nonC^P37 mutant at any of the four light intensities tested. An independently isolated nonC mutant allele, nonC^MC45, showed a similar, selective synaptic impairment. This mutant also had no detectable photoreceptor synaptic on-transients, and displayed off-transients of reduced amplitude. The peak phototransduction responses of photoreceptors were slightly depressed in nonC^P37 and comparable with the wild type in nonC^MC45. The loss of photoreceptor synaptic transmission in both nonC mutant alleles was totally rescued by introduction of the wild-type candidate gene, demonstrating that the synaptic defect is due solely to the loss of this single gene function (Long, 2010).

Candidates for the nonC gene were identified by a microarray-based approach screening for statistically significant alterations in mRNA levels of genes within the chromosomally mapped mutant interval. Deficiency mapping using ERGs to examine resulting photoreception phenotypes to determine whether complementation had occurred placed nonC between the right breakpoint of deficiency Df(1)ED6849 and right breakpoint of duplication Dp(1,Y)dx49. To accommodate possible mismatch between reported chromosomal breakpoints and gene locations, microarray data were examined in a wider region than that defined by mapping; namely 6C13-6E2, the 120 kb region between coordinates X:6660000 and X:6780000, which contains 17 genes. Microarray data showed that four genes showed statistically significant changes in mRNA levels in nonC mutants: shf, Smg1, CG4557 and CG4558. Thus, these genes were identified as candidates (Long, 2010).

All genes were fully sequenced in three different nonC mutants; nonC^P37, nonC^MC45 and nonC^MC47. No mutations were found in the shf, CG4557 or CG4558 genes in any nonC mutants. By contrast, the Smg1 gene carried a T to C transition in nucleotide 2552, resulting in the isoleucine to threonine change in residue 851 in both nonC^MC45 and nonC^MC47. These results suggested that Smg1 was the most likely candidate for the nonC gene. Following this identification, previously described Smg1 alleles were obtained, including Smg1^rst2, a premature stop codon resulting at residue 704 AAA to TAA (Chen, 2005), and Smg1^32AP, a genetic null as a result of a point mutation in the Smg1 kinase domain at residue 1651 (Metzstein, 2006; Long, 2010).

The most definitive proof of gene identity is to introduce the candidate wild-type gene into the mutant and assay rescue of mutant phenotype. A genomic Smg1 construct driven by the native promoter was used to ensure proper spatiotemporal expression. The Smg1 gene is ~13.6 kb and the predicted promoter region is ~3.2 kb, generating a sequence of nearly 17 kb and making the use of P-element-mediated transformation questionable. Therefore P[acman]-mediated recombination and ΦC31-mediated genome integration were used. Insertion of the genomic construct in several Smg1 mutant backgrounds was confirmed by PCR. Two copies of wild-type Smg1 completely rescued the nonC synaptic phenotype in the adult visual system and larval NMJ in both the nonC^P37 and nonC^MC45 mutant alleles. These results together strongly support the conclusion that nonC and Smg1 are the same gene. Drosophila Smg1 encodes PIKK, with 34.1% protein sequence identity between CG32743 and human SMG1 (Chen, 2005; Long, 2010).

Smg1 is a key regulatory kinase in the NMD pathway. NMD is an mRNA surveillance system that is critical for maintenance of transcript integrity. The Smg proteins (Smg1–Smg7) act together to degrade mRNA transcripts containing nonsense mutations that would produce truncated proteins with potentially deleterious activities. This study reveals that Smg1 functions to protect the development of synaptic morphological architecture and maintain high-fidelity neurotransmission function, especially under conditions of heightened activity. Among isolated Smg mutants, only Smg1 nulls are viable as adults; Smg5 mutants die as first instars, and Upf2 (Smg3) and Smg6 die as pupae. These lethal periods have restricted the adult analyses to Smg1 mutants, and larval analyses to Smg1, Smg3 and Smg6 mutants, and might indicate that Smg1 is less essential for NMD than the other complex components. Together, these studies indicate that NMD activity is required in at least two disparate synaptic classes, retinal photoreceptor synapses and neuromuscular junction, suggesting that an NMD-mediated protective mechanism is broadly important in synapses throughout the nervous system (Long, 2010).

A great deal of effort has gone into exploring the roles of mRNA regulation in synaptic mechanisms. In particular, mechanisms of mRNA stabilization, trafficking, RISC-mediated degradation and localized translation have all been recently determined to occur in proximity to synapses and/or with important roles in synapse regulation. These insights have raised the question of possible NMD involvement (Giorgi, 2007). This study reports that disruption of NMD-mediated mRNA regulation results in impaired morphological NMJ development, loss of functional transmission at central (histaminergic) synapses and peripheral (glutamatergic) synapses, and impaired synaptic vesicle cycling at the NMJ, especially under conditions of elevated demand. These results indicate that NMD has an important role in maintaining synapse architecture and high-fidelity synaptic efficacy (Long, 2010).

Synaptic morphological defects can be readily observed with the loss of appropriate mRNA regulation of synaptic components. This requirement has been characterized for a number of mRNA-binding proteins, prominently including the fragile-X mental retardation protein (FMRP). This study shows that loss of mRNA regulation via disruption of the NMD pathway also impairs synaptic architecture at the well-characterized Drosophila NMJ. In Smg1 mutants, NMJ synaptic arbors appear underdeveloped and underelaborated, with limited differentiation outside the immediate vicinity of the initial point of muscle innervation. The range of morphological phenotypes includes overall reduction in synaptic terminal area, decrease in synaptic arbor branching by ~50% and decrease in synaptic bouton number by ~50% (Long, 2010).

Defects in NMD mutants are presumably caused by the production of truncated proteins with dominant-negative or deleterious gain-of-function activities that damage the machinery of synaptic development. It is proposed that Smg mutants are unable to prevent translation of damaged transcripts, and thus the production of 'toxic proteins' results in smaller, less-developed and functionally compromised synapses. Where and when the NMD machinery performs this essential activity is uncertain; it could be wholly in the neuronal soma, or locally at synapses for transcripts that might undergo local translation. It is noted, however, that the Smg1 mutant defects revealed in this study appear largely restricted to the presynaptic domain; postsynaptic scaffolding and glutamate receptor expression is not detectably altered, and there is no detectable change in postsynaptic physiological function. Local mRNA translation in growth cones has been demonstrated to be crucial for axon pathfinding and synapse maturation. The defects reported in this study could occur in the motor neuron growth cone or during later stages of synaptic maturation (Long, 2010).

The cyclic events that orchestrate the release of neurotransmitter at the synapse consist of a rapidly repeating series of vesicular trafficking, exocytosis and endocytosis steps. There are many molecular stages in this pathway that are susceptible to disruption, which when impaired cause similar defects in vesicle cycling. Consistent with a protective mechanism for the NMD pathway in this mechanism, experimental introduction of peptide fragments of synaptic vesicle cycle proteins is a well-established means of investigating molecular requirements. The multiple Smg gene mutants analyzed in this study all show profound disruptions in the endocytosis phase of the synaptic vesicle cycle, revealing an impairment in the ability to retrieve vesicular membrane and associated proteins from the plasma membrane after vesicle fusion. Moreover, disruption of the NMD pathway causes a significantly reduced functional readily releasable pool of vesicles, although the morphological releasable pool appears relatively intact in Smg1 mutants after periods of both basal and intense synaptic activity. This suggests that the loss of neurotransmission in Smg mutants is due not only to impaired vesicle biogenesis, but perhaps also to the interruption of molecular functions underlying evoked vesicle exocytosis (Long, 2010).

Many proteins have demonstrated roles in synaptic vesicle endocytosis and exocytosis. Loss of NMD probably generates 'toxic protein fragments' that interfere with many of these proteins, both individually and by preventing the formation of presynaptic molecular complexes. One of the most predicted consequences of such fragmentary proteins is to act as dominant negatives by interfering with (or out-competing) appropriate protein-protein interactions during complex formation. The Smg mutants show a more-severe impairment of presynaptic function under conditions of high-frequency stimulation. During such periods of elevated demand, the stress placed on the synaptic vesicle machinery is obviously greater, demanding that proteins function more rapidly and that molecular complexes form and function at a faster rate. This model predicts that mutant proteins produced in the absence of the NMD pathway would therefore have a greater deleterious impact under conditions of high-frequency stimulation. The inhibition caused by truncated synaptic vesicle proteins presumably accounts for the very small amount of vesicle cycling evident at the Smg mutant synapse during periods of high demand (Long, 2010).

It was recently shown that loss of the NMD-initiating factor and exon-junction complex component eIF4AIII leads to synaptic functional impairments (Bramham, 2008; Giorgi, 2007). It is also known that localized mRNAs encode receptors, cytoskeletal proteins and regulatory proteins that are critical for synaptic development, and that the localized translational regulation of these mRNAs is crucial for synaptic stability and function . Future work will focus on the identification of the mRNA species encoding synaptic proteins that accumulate in the absence of the NMD pathway, mRNAs locally translated in the late-maturing axonal growth cone and potentially presynaptic terminal, and study of how the production of aberrant proteins synthesized in the absence of NMD activity impairs synaptic architecture and presynaptic function (Long, 2010).

Functions of the nonsense-mediated mRNA decay pathway in Drosophila development

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. This study describes the isolation and characterization of Drosophila 'photoshop' mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, the PI3K-related kinase Smg1 was found to potentiate but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, the SV40 3' UTR, present in many Drosophila transgenes, was found to target the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells (Metzstein, 2006).

Genetic analysis demonstrated a striking difference in the developmental requirements of Smg1 compared to those of Upf1 and Upf2. First, Upf1 and Upf2 are essential genes, whereas an amorphic Smg1 allele resulted in viable and fertile animals. Second, a Upf2 mutation abolished NMD of an Adh PTC allele, whereas the amorphic Smg1 mutation only modestly reduced NMD efficiency. Third, the magnitude of the Smg1 mutant effect differed at different targets. At some targets, such as oda, there was little or no effect of the Smg1 mutation, whereas at other targets, such as tra and a GFP transgene, the Smg1 mutant effect was up to half that of the Upf2 mutant. The small and gene-selective effect of Smg1 could explain why a recent genetic analysis failed to detect a role for Drosophila Smg1 in NMD (Chen, 2005), whereas earlier Drosophila cell culture studies suggested an important role for the gene (Gatfield, 2004). The small and gene-selective function of Drosophila Smg1 contrasts with genetic results in C. elegans, which did not identify differences in the requirements of smg-1 and the Upf1 and Upf2 homologs smg-2 and smg-3 (Hodgkin, 1989). One possibility is that Drosophila has another protein with activity similar to that of Smg1. This seems unlikely because Smg1 is the only sequence ortholog of Smg1-family genes in the Drosophila genome, although there are other genes that encode proteins with PI3K-related kinase domains. Another possibility is that phosphorylation of Upf1 by Smg1 is not absolutely required for Upf1 activity in Drosophila but only enhances its activity or reactivates spent protein after a catalytic cycle. This would be more similar to the NMD pathway in yeast, which lacks a Smg1 ortholog and is thought to function without a Upf1 phosphorylation cycle, than to the NMD pathways in C. elegans and vertebrates, where the Upf1 phosphorylation cycle is thought to be essential for pathway activity (Metzstein, 2006).

Search PubMed for articles about Drosophila Smg1

Barbee, S. A., et al. (2006). Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron 52: 997-1009. PubMed ID: 17178403

Bramham, C. R., Worley, P. F., Moore, M. J. and Guzowski, J. (2008). The immediate early gene arc/arg3.1: regulation, mechanisms, and function. J. Neurosci. 28: 11760-11767. PubMed ID: 19005037

Chen, Z., Smith, K. R., Batterham, P. and Robin, C. (2005). Smg1 nonsense mutations do not abolish nonsense-mediated mRNA decay in Drosophila melanogaster. Genetics 171: 403-406. PubMed ID: 15965240

Giorgi, C., et al. (2007). The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression. Cell 130: 179-191. PubMed ID: 17632064

Gatfield, D. and Izaurralde, E. (2004). Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429: 575-578. PubMed ID: 15175755

Hodgkin, J., Papp, A., Pulak, R., Ambros, V. and Anderson, P. (1989). A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics 123: 301-313. PubMed ID: 2583479

Metzstein, M. M. and Krasnow M. A. (2006). Functions of the nonsense-mediated mRNA decay pathway in Drosophila development. PLoS Genet. 2: e180. PubMed ID: 17196039

Long, A. A., et al. (2010). The nonsense-mediated decay pathway maintains synapse architecture and synaptic vesicle cycle efficacy. J. Cell Sci. 2010 Oct 1;123(Pt 19): 3303-15. PubMed ID: 20826458

Shibuya, T., Tange, T., Sonenberg, N. and Moore, M. J. (2004). eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 11: 346-351. PubMed ID: 15034551

Shibuya, T., Tange, T., Stroupe, M. E. and Moore, M. J. (2006). Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay. RNA 12: 360-374. PubMed ID: 16495234

Waung, M. W., Pfeiffer, B. E., Nosyreva, E. D., Ronesi, J. A. and Huber K. M. (2008). Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate. Neuron 59: 84-97. PubMed ID: 18614031

date revised: 2 June 2012

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