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Alternative Splicing of Drosophila Nmnat Functions As a Switch to Enhance Neuroprotection Under Stress

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Alternative Splicing of Drosophila Nmnat Functions As a Switch to Enhance Neuroprotection Under Stress ARTICLE Received 9 Dec 2014 | Accepted 28 Oct 2015 | Published 30 Nov 2015 DOI: 10.1038/ncomms10057 OPEN Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress Kai Ruan1,2, Yi Zhu1,2,*, Chong Li1,3,*, Jennifer M. Brazill1,2 & R Grace Zhai1,2,3 Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuro- protective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. Here we show that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. Our results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection. 1 Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA. 2 Program in Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA. 3 Program in Human Genetics and Genomics, University of Miami Miller School of Medicine, Miami, Florida 33136, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.G.Z. (email: [email protected]). NATURE COMMUNICATIONS | 6:10057 | DOI: 10.1038/ncomms10057 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10057 icotinamide mononucleotide adenylyltransferase functions and biochemical and cellular properties of DmNMNAT (NMNAT) is a highly conserved housekeeping enzyme isoforms, and directly monitored the shift in alternative Ncatalysing reversibly the last step in the NAD salvage splicing in vivo in the Drosophila nervous system using an pathway in all living organisms1. In addition, NMNAT proteins alternative-splicing reporter. Our results identify alternative are essential for neuronal maintenance and protection2. splicing of DmNmnat as a switch for regulating neuroprotection, Functional studies in Drosophila and mammals have shown and reveal a neuronal mechanism of achieving NMNAT- that loss of NMNAT causes neurodegeneration. For example, loss mediated self-protection under stress. of DmNmnat in photoreceptors causes retinal degeneration2; loss-of-function mutations in human Nmnat1 cause Leber congenital amaurosis 9 (LCA9)3–6, a childhood blindness Results disease that involves degeneration of the photoreceptors3,5,7; DmNmnat is alternatively spliced. Drosophila melanogaster has and loss of mouse Nmnat2 causes axonal degeneration8. one Nmnat gene that contains seven tandem exons (Fig. 1a). The neuronal maintenance function of NMNAT can be Gene structure analyses suggest that DmNmnat is alternatively enhanced by overexpression to achieve neuroprotection against spliced into two mRNA variants RA and RB, with the first four a variety of neurodegenerative conditions9. For example, overexpression of yeast NMNAT (NMA1) protects against a 24,945 K 24,946 K 24,947 K polyglutamine (polyQ)-induced cytotoxicity10, expression of 3R Box 1 Box 2 Drosophila NMNAT protects against light-induced retinal Drosophila Nmnat GATACGTACACTGA TCAGTGTGCGTATC 2 degeneration or Wallerian degeneration in cultured rat dorsal Gene 1234567 root ganglion neurons11 and ectopic expression of any of the E1F E5R E7R three human NMNAT confers neuroprotection against axonal mRNA RA RB degeneration in cultured neurons or transgenic mouse models PA (see reviews9,12). These observations suggest that the CDS PB neuroprotective property of NMNAT is conserved through PC PD distant phyla. In most of the mammalian degenerative models, particularly in axonal degeneration, it has been suggested that b c Nmnat –/– F E1F yw Actin-GAL4>UAS-HA- the NAD synthetic activity of NMNAT is required for Primers: kDa 12,13 R E7R E5R 22 °C 37 °C –PC –PD IB: DmNMNAT its neuroprotective activity (see reviews ). In addition to its (kb)10 75 (n/s) enzymatic function, work from our group14 and others10,15 has 3 50 PA identified a chaperone function of DmNMNAT that contributes PC HA-PC 1.5 37 to neuroprotection by directly interacting with unfolded/ 1 RA RB PB HA-PD PD misfolded proteins in the nervous system, reducing the load of 0.5 25 toxic protein oligomers and facilitating the clearance of unfolded 14,16,17 proteins . These studies in different model systems have d +/+ –/– provided evidence supporting two roles of NMNAT in neurons: Nmnat Nmnat Actin-GAL4 Wild type (yw) chaperone and NAD metabolism. It remains unclear whether, UAS-PC UAS-PD and if so how, neurons regulate the partitioning of NMNAT into these two roles and balance metabolic and self-protective activities. Alternative splicing is an important post-transcriptional regula- tory mechanism for cells to generate transcript variability and 18,19 NMNAT proteome diversity . It is estimated that 95% of multiexonic nc82 genes in humans and 65% in Drosophila are alternatively DAPI spliced20,21. In the nervous system, this process is especially common for gene regulation. Recent work has identified several aspects of neural development and function that are specifically controlled by alternative splicing. Examples include fibroblast growth factor 8 in neural tube patterning22,theDrosophila homologue of Down’s syndrome cell-adhesion molecule in the NMNAT axon guidance and dendrite morphogenesis23–25, as well as neurexins and neuroligins in synaptogenesis26,27. In the mature Figure 1 | DmNmnat is alternatively spliced into two major mRNA nervous system, it has been shown that Nova-1 regulates neuronal- variants and possibly four protein isoforms. (a) Diagram of gene structure specific alternative splicing and is essential to maintain neuronal of DmNmnat and predicted expression products. Boxes 1 and 2 mark the health28, and neuronal activity dynamically regulates the splicing of sequences that form the stem–loop structure that is required for alternative products that determine neuronal excitation such as SNARE splicing34. Black arrows indicate the sequence locations of forward (E1F) protein SNAP25 (ref. 29). Misregulation of alternative splicing or and reverse (E5R, E7R) primers used. Red arrows mark the translation start of splicing regulatory factors can lead to multiple human sites. (b) RT–PCR of total RNA from head extracts of wild-type flies (yw) neurodegenerative diseases30, such as spinal muscular atrophy31, using two primer sets as indicated in a.(c) Western blot analysis using a frontotemporal lobar dementias and amyotrophic lateral sclerosis32, polyclonal DmNMNAT antibody2 of head extracts from wild-type flies (yw) and tauopathies33. These studies highlight the important role of under 22 and 37 °C, or flies overexpressing PC and PD isoforms with alternative splicing in nervous system development and function. ubiquitous actin-GAL4 driver in Nmnat À / À null background. Grey However, the role of alternative splicing in neuronal maintenance arrowheads indicate likely nonspecific bands. (d) Adult fly brains from wild- and protection has not been as widely studied. type (yw)orNmnat À / À null flies expressing PC or PD were probed for Here we seek to address the mechanisms of how neurons NMNAT (green), synapse marker nc82 (magenta) and DAPI (blue). Bottom regulate the expression of DmNmnat to enhance its neuropro- row shows the respective NMNAT channel of the boxed area in top row. tective capability. We have characterized the neuroprotective Scale bars, 20 mm for each row. 2 NATURE COMMUNICATIONS | 6:10057 | DOI: 10.1038/ncomms10057 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10057 ARTICLE exons constitutively included and the last three exons expressed adult brains showed a nonoverlapping pattern, where differentially spliced (Fig. 1a). A previous report analysing splice PC is highly enriched in the cell body and PD is predominantly sites in the Drosophila genome identified sequences in the fourth cytoplasmic (Fig. 1d and Supplementary Fig. 2). Therefore, the intron and fifth exon that can form a ‘stem–loop’ structure to endogenous DmNMNAT expression pattern is a combined facilitate the alternative splicing event (Boxes 1 and 2 in Fig.1a)34. distribution patterns of all isoforms. These data suggest that In addition, the first exon contains two putative translational start alternative splicing of DmNmnat produces two protein products codons, which may further increase the protein diversity. that localize to distinct subcellular compartments: nuclear PC and To determine the endogenous DmNmnat variants, we first cytosolic PD. used variant-specific amplification primers located in exon 5 To further investigate the mechanism of DmNMNAT (E5R) and 7 (E7R) to identify the mRNA transcripts. Two mRNA localization, we analysed the C-terminal amino-acid sequence transcripts from adult fly brain extracts were identified (Fig. 1b) of PC and PD and identified a putative nuclear localization
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