Skip to main content

Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte



Spinal muscular atrophy (SMA) results from insufficient levels of the survival motor neuron (SMN) protein. Drosophila is conducive to large-scale genetic-modifier screens which can reveal novel pathways underpinning the disease mechanism. We tested the ability of a large collection of genomic deletions to enhance SMN-dependent lethality. To test our design, we asked whether our study can identify loci containing genes identified in previous genetic screens. Our objective was to find a common link between genes flagged in independent screens, which would allow us to expose novel functions for SMN in vivo.


Out of 128 chromosome deficiency lines, 12 (9.4%) were found to consistently depress adult viability when crossed to SMN loss-of-function heterozygotes. In their majority, the enhancing deletions harboured genes that were previously identified as genetic modifiers, hence, validating the design of the screen. Importantly, gene overlap allowed us to flag genes with a role in post-transcriptional regulation of mRNAs that are crucial for determining the axes of the oocyte and future embryo. We find that SMN is also required for the correct localisation of gurken and oskar mRNAs in oocytes. These findings extend the role of SMN in oogenesis by identifying a key requirement for mRNA trafficking.


Spinal muscular atrophy (SMA) is a motor neuron disease caused by homozygous mutations in the survival motor neuron 1 (SMN1) gene that are partially compensated by the paralogous SMN2 gene. SMA patients have insufficient levels of the SMN protein, a situation triggering lower motor neuron degeneration and profound muscle weakness that restricts mobility and, in severe cases, results in respiratory failure and death [1]. SMN operates as part of a large multiprotein complex whose constituents also include Gemins 2–8 and Unrip [2]. The SMN complex is known to chaperone the assembly of ribonucleoproteins (RNPs) including small nuclear RNPs (snRNPs), which form the core components of the spliceosome [3], and messenger RNPs (mRNPs), which ensure transport as well as cytosolic localisation of mRNAs [4]. Whether either or both RNP assembly reactions are perturbed in SMA remains unclear. Animal models including the fruit fly Drosophila melanogaster are key for exploring the in vivo function of the SMN protein (reviewed in [5]). To this end, SMA-causing missense mutations (SMN73Ao) or deletion of the fly SMN gene orthologue leads to motor dysfunction in addition to defective neuromuscular junction (NMJ) morphology and transmission [6,7,8].

Drosophila is conducive to large-scale genetic-modifier screens which can potentially reveal novel pathways involved in the disease mechanism. The first Drosophila SMN genetic screen assessed whether a collection of transposon-induced mutations either enhanced or suppressed the lethality of SMN73Ao heterozygotes and homozygotes, respectively. The identified modifier genes had no obvious role in RNP assembly with some including components of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signalling pathway [7, 9]. In a later study, the same lab performed a complementary screen this time using a hypomorphic SMN RNAi allele to increase sensitivity. A larger number of candidate genes that function in various pathways including RNA metabolism were successfully discovered [10]. Aiming at performing an independent SMN genetic screen, we tested the ability of a large collection of genomic deletions to reduce the viability of SMN73Ao heterozygotes. To test our design, we asked whether our study can expose genomic regions containing genes identified in previous genetic screens. Finally, by exploring a common link between genes flagged in independent screens, we expose a function for SMN in post-transcriptional mRNA regulation in vivo.

Main text


Fly stocks

Flies were cultured on standard molasses/maizemeal and agar medium in plastic vials at an incubation temperature of 25 °C. The SMN73Ao mutant has been characterised previously [6, 7, 11,12,13,14]. The chromosome 2 and 3 deficiency lines were obtained from the Bloomington Drosophila stock center at Indiana University, USA.

Genetic screen

Deficiency lines were crossed to the SMN73Ao mutant line to determine whether haploinsufficiency of genomic regions have a negative influence on the adult viability of SMN73Ao heterozygotes. Adult viability was calculated as the percentage number of adult flies eclosed divided by the expected number for the cross. For deficiencies that were found to depress adult viability, the cross was repeated for confirmation.


Genes mapped within the SMN73Ao-interacting chromosome deficiencies were listed using the ‘CytoSearch’ query tool on FlyBase [15] (; FB2017_02 release). The ‘HitList’ tool was applied to the gene set to analyse the frequencies of values for gene ontology (GO) controlled vocabulary (CV) terms for biological process. GO enrichment analysis using the PANTHER classification system was performed using the enrichment analysis tool on the gene ontology consortium (GOC) website (

Generation of mutant germline clones

The FLP-DFS (yeast flippase-dominant female sterile) technique (reviewed in [16]) was utilized to generate SMN73Ao mutant germline clones. Virgin females having the w; SMN73Ao FRT2A/TM3, Ser genotype were crossed to y w hsFLP; ovoD1 FRT2A/TM3, Ser males and recombination between the FRT (flippase recombinase target) sites in the resulting progeny was stimulated through heat-shock at 37 °C for 1 h at day 3, 4, and 5 after egg hatching. Egg chambers that survive beyond stage 4 in the ovaries of the female offspring (y w hsFLP; SMN73Ao FRT2A/ovoD1 FRT2A) lack ovoD1 and are hence homozygous for SMN73Ao.

In situ hybridization

Ovaries were dissected in PBS (phosphate buffered saline) and later fixed in 4% paraformaldehyde in PBS at room temperature. Following treatment with proteinase K, ovaries were washed in PBS + 0.1% Tween20, re-fixed and washed again. They were later washed in a 1:1 solution formed of PBS + 0.1% Tween20: hybridization buffer (50% deionized formamide, 5× saline sodium citrate, 100 μg/ml E. coli tRNA, 50 μg/ml heparin, and 0.1% Tween20 in DEPC-water). Following pre-hybridisation for at least 1 h at 55 °C in hybridization buffer, DIG-labelled antisense gurken or oskar RNA probes were allowed to hybridise overnight in the same conditions. Three washing steps at 65 °C using (a) hybridisation buffer, (b) 1:1 PBS + 0.1% Tween20: hybridisation buffer, and (c) PBS + 0.1% Tween20 in that order, preceded incubation with sheep anti-DIG HRP-coupled antibody (1:2000; Roche Diagnostics Ltd.) for 2 h at room temperature. The hybridisation signal was amplified with Cy3-tyramide (PerkinElmer) and the ovaries were counterstained with Hoechst 33342 prior to mounting. Confocal images captured using the oil 40× magnification objective were processed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Based on oocyte size and distinct mRNA localisation patterns, assessment was restricted to early stage 10 egg chambers.


To gain insights on pathways involved in SMA, we attempted at conducting a pilot genetic screen using part of the Bloomington Deficiency Kit. The Kit provides maximal coverage of the Drosophila genome with a minimal number of molecularly-defined deletions, hence facilitating genome-wide genetic screens [17]. Our screen involved a single stage designed to identify deletions that induced a pronounced decrease in adult viability when placed within an SMN loss-of-function heterozygous background. Previous studies suggested a strong association between the degree of adult viability and motor dysfunction phenotypes [7]. SMN73Ao/TM6B virgin females were mated to males carrying deletions spanning either arm of chromosome 3 (3R/3L) or the left arm of chromosome 2 (2L), hence, targeting approximately 50% of the Drosophila genome. In the F1 generation, flies of the appropriate genotype were identified to determine whether deletions placed in trans with the SMN73Ao chromosome induced reduced viability compared to flies having the SMN73Ao chromosome only (Fig. 1). The percentage number of flies eclosed was calculated and deletions were defined as ‘enhancers’ if they induced ≥ 15% difference, with the interaction strength being classified as mild (+, ≥ 15%), moderate (++, ≥ 25%), strong (+++, ≥ 35%) or intense (++++, ≥ 45%).

Fig. 1
figure 1

Schematic representation of the genetic screen. Individual second or third chromosome deficiencies were introduced in flies that are heterozygous for the SMN73Ao loss-of-function allele. In the F1 generation, enhancing deletions were identified as those that reduced significantly the percentage number of flies eclosed when in trans with the SMN73Ao chromosome

In total, 128 chromosome deficiency lines were evaluated and 12 (9.4%) were found to consistently depress the viability of SMN mutant heterozygotes (Table 1; Additional file 1: Table S1). The Df(3L)81k19 deletion on the third chromosome produced the strongest enhancement, thereby leading to no adult viable flies. This was expected since one of the genes covered by the deficiency is the SMN gene, hence, Df(3L)81k19 unsurprisingly failed to complement the loss-of-function SMN73Ao mutation. Systematic evaluation of the candidate genes located within the genomic intervals flagged by the enhancing deletions is a laborious endeavour without a guarantee of success considering that more than one gene might be responsible for the enhanced phenotype. We therefore generated a ‘HitList’ formed of the genes uncovered by the enhancing deletions and probed the gene set for GO enrichment. Results were not statistically significant but some of the most frequent GO terms for biological process are pathways known to be disrupted in motor neuron disease including oxidation–reduction, neurogenesis, proteolysis, transcription, and translation [18] (Additional file 2: Table S2).

Table 1 Chromosome deficiency lines that depress the viability of SMN7Ao heterozygotes

Interestingly, all the identified deletions with the exception of one (Df[2L]ed1), harboured genes that were previously found to modify SMN mutant phenotypes [7, 10]. In addition to validating the design of our screen, this finding can potentially flag genetic loci that overlap independently-conducted genetic screens. In this regard, we found a common thread running through 3 enhancing deletions. Each cover a previously identified genetic modifier that is known to have a role in post-transcriptional regulation of mRNAs that are crucial for determining the axes of the oocyte and future embryo [19]. The genes include encore (enc) covered by Df(3L)HR119, Syncrip (Syp) covered by Df(3R)BSC43, and hephaestus (heph) covered by Df(3R)B81 (Table 1). Specifically, either gene was found to be required for the localisation of gurken and/or oskar mRNAs in oocytes [20,21,22]. Notably, considering the gene set uncovered by our genetic screen, oogenesis was also identified as one of the top-ranked most-frequent GO terms for biological process (Additional file 2: Table S2). The studies that have thus far explored a role for SMN in oogenesis have been few. Lee et al. [11] showed that defective nuclear organisation was the most prominent early defect in SMN mutant Drosophila eggs. We have previously observed similar phenotypes in egg chambers mutated for the SMN-associated DEAD-box helicase, Gemin3 [23, 24]. Considering our assessment of the genetic screen results, we asked whether SMN is also required for the correct localisation of gurken and oskar mRNAs. To this end, we find that in SMN73Ao mutant oocytes, gurken mRNA was partially mis-localised, with transcript localisation skewed towards with the anterior or dorsal side (Fig. 2). This is in contrast to control oocytes in which gurken mRNA was always found tightly localised in a dorsal-anterior cap above the oocyte nucleus. Localisation of oskar mRNA was also defective. By the end of stage 8 of oogenesis, oskar mRNA accumulates in a crescent that is tightly localised to the posterior of the oocyte. In SMN73Ao mutant oocytes, posterior oskar mRNA was only faintly detected (Fig. 2). Overall, these results extend the role of SMN in oogenesis by identifying a requirement for mRNA localisation.

Fig. 2
figure 2

Aberrant mRNA localisation in SMN73Ao mutant oocytes. Stage 10 egg chambers hybridised by either gurken or oskar antisense RNA probes and counterstained for DNA. Top is posterior whereas right is dorsal. In the top panel, arrows mark the oocyte nucleus; in the bottom panel, the arrow head marks residual transcript sometimes detected at the anterior corner


In vivo studies have been supportive of a role for the SMN complex in snRNP assembly, hence, disturbances in this pathway and the consequential transcriptome abnormalities are thought to be the primary drivers of the progressive neuromuscular degeneration underpinning SMA (reviewed in [3]). In particular, we have previously shown that, in Drosophila, perturbation of snRNP assembly factors results in motor defects that mirror those described on loss of SMN or the Gemin constituents of the SMN complex [25,26,27,28]. Here, we exploited the genetic tractability of the fly system to identify genetic loci that influence SMN activity, thereby aiming at uncovering novel insights on SMN function in vivo. Thorough mining of the gene set uncovered by the SMN lethality-enhancing deletions allowed us to flag genes with a common function in RNA transport that were ‘hits’ in previous genetic screens. Making use of the extensively-studied Drosophila ovary, these findings led us to show that RNA transport is defective in SMN mutant oocytes. Although such phenotypes do not exclude a role for SMN in snRNP assembly, our results provide in vivo evidence implicating a function for SMN in RNA transport. This is corroborated by in vitro studies that are indicative of an involvement of SMN in mRNA trafficking within neurons (reviewed in [4, 29]).

Our study also extends the requirement of SMN during oogenesis. Hence, in addition to nuclear organisation and maintenance of the structural integrity of RNP bodies [11, 30], SMN is also crucial for the cytoplasmic localisation of mRNA transcripts that specify the future embryonic body axes. It is highly likely that the evident mislocalisation of gurken and oskar mRNAs contribute to the embryonic death observed for oocytes derived from an SMN mutant germline [6]. Our findings corroborate those by Grice and Liu [13] who showed that SMN73Ao homozygous mutant neuroblasts failed to correctly localise the RNP component Miranda at the basal membrane. The exact function of SMN in mRNA trafficking remains unclear. Similar to its role in snRNP assembly, SMN might act as a molecular chaperone for the assembly of mRNP complexes [31]. The Drosophila ovary can however serve as a model system to further investigate the in vivo function of SMN in mRNA transport and localisation. Such studies can potentially provide insights on parallel activities occurring within the neuromuscular system and whose perturbation can lead to SMA.


Limitations arise from the lack of systematic evaluation of all the candidate genes covered by the enhancing deletions. In this regard, the contribution of previously identified genetic modifiers to the enhancing effect of the deletions is tentative.



gene ontology


spinal muscular atrophy


survival motor neuron


messenger ribonucleoprotein




small nuclear ribonucleoprotein


  1. Burghes AH, Beattie CE. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci. 2009;10:597–609.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Cauchi RJ. SMN and Gemins: ‘we are family’… or are we? Insights into the partnership between Gemins and the spinal muscular atrophy disease protein SMN. BioEssays. 2010;32:1077–89.

    Article  PubMed  CAS  Google Scholar 

  3. Lanfranco M, Vassallo N, Cauchi RJ. Spinal muscular atrophy: from defective chaperoning of snRNP assembly to neuromuscular dysfunction. Front Mol Biosci. 2017;4:41.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Donlin-Asp PG, Bassell GJ, Rossoll W. A role for the survival of motor neuron protein in mRNP assembly and transport. Curr Opin Neurobiol. 2016;39:53–61.

    Article  PubMed  CAS  Google Scholar 

  5. Aquilina B, Cauchi RJ. Modelling motor neuron disease in fruit flies: lessons from spinal muscular atrophy. J Neurosci Methods. 2018.

    Article  PubMed  Google Scholar 

  6. Chan YB, Miguel-Aliaga I, Franks C, Thomas N, Trulzsch B, Sattelle DB, Davies KE, van den Heuvel M. Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum Mol Genet. 2003;12:1367–76.

    Article  PubMed  CAS  Google Scholar 

  7. Chang HC, Dimlich DN, Yokokura T, Mukherjee A, Kankel MW, Sen A, Sridhar V, Fulga TA, Hart AC, Van Vactor D, et al. Modeling spinal muscular atrophy in Drosophila. PLoS ONE. 2008;3:e3209.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Imlach WL, Beck ES, Choi BJ, Lotti F, Pellizzoni L, McCabe BD. SMN is required for sensory-motor circuit function in Drosophila. Cell. 2012;151:427–39.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Sen A, Yokokura T, Kankel MW, Dimlich DN, Manent J, Sanyal S, Artavanis-Tsakonas S. Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling. J Cell Biol. 2011;192:481–95.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Sen A, Dimlich DN, Guruharsha KG, Kankel MW, Hori K, Yokokura T, Brachat S, Richardson D, Loureiro J, Sivasankaran R, et al. Genetic circuitry of survival motor neuron, the gene underlying spinal muscular atrophy. Proc Natl Acad Sci USA. 2013;110:E2371–80.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lee L, Davies SE, Liu JL. The spinal muscular atrophy protein SMN affects Drosophila germline nuclear organization through the U body-P body pathway. Dev Biol. 2009;332:142–55.

    Article  PubMed  CAS  Google Scholar 

  12. Rajendra TK, Gonsalvez GB, Walker MP, Shpargel KB, Salz HK, Matera AG. A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J Cell Biol. 2007;176:831–41.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Grice SJ, Liu JL. Survival motor neuron protein regulates stem cell division, proliferation, and differentiation in Drosophila. PLoS Genet. 2011;7:e1002030.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Lee S, Sayin A, Cauchi RJ, Grice S, Burdett H, Baban D, van den Heuvel M. Genome-wide expression analysis of a spinal muscular atrophy model: towards discovery of new drug targets. PLoS ONE. 2008;3:e1404.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Gramates LS, Marygold SJ, Santos GD, Urbano JM, Antonazzo G, Matthews BB, Rey AJ, Tabone CJ, Crosby MA, Emmert DB, et al. FlyBase at 25: looking to the future. Nucleic Acids Res. 2017;45:D663–71.

    Article  PubMed  CAS  Google Scholar 

  16. Cauchi RJ, van den Heuvel M. The fly as a model for neurodegenerative diseases: is it worth the jump? Neurodegener Dis. 2006;3:338–56.

    Article  PubMed  Google Scholar 

  17. Cook RK, Christensen SJ, Deal JA, Coburn RA, Deal ME, Gresens JM, Kaufman TC, Cook KR. The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome. Genome Biol. 2012;13:R21.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Robberecht W, Philips T. The changing scene of amyotrophic lateral sclerosis. Nat Rev Neurosci. 2013;14:248–64.

    Article  PubMed  CAS  Google Scholar 

  19. Lasko P. mRNA localization and translational control in Drosophila oogenesis. Cold Spring Harb Perspect Biol. 2012;4:a012294.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Hawkins NC, Van Buskirk C, Grossniklaus U, Schupbach T. Post-transcriptional regulation of gurken by encore is required for axis determination in Drosophila. Development. 1997;124:4801–10.

    PubMed  CAS  Google Scholar 

  21. McDermott SM, Meignin C, Rappsilber J, Davis I. Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification. Biol Open. 2012;1:488–97.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Besse F, Lopez de Quinto S, Marchand V, Trucco A, Ephrussi A. Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation. Genes Dev. 2009;23:195–207.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Cauchi RJ. Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis. BMC Res Notes. 2012;5:120.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Curmi F, Cauchi RJ. The multiple lives of DEAD-box RNA helicase DP103/DDX20/Gemin3. Biochem Soc Trans. 2018;46:329–41.

    Article  PubMed  CAS  Google Scholar 

  25. Borg RM, Fenech Salerno B, Vassallo N, Bordonne R, Cauchi RJ. Disruption of snRNP biogenesis factors Tgs1 and pICln induces phenotypes that mirror aspects of SMN-Gemins complex perturbation in Drosophila, providing new insights into spinal muscular atrophy. Neurobiol Dis. 2016;94:245–58.

    Article  PubMed  CAS  Google Scholar 

  26. Lanfranco M, Cacciottolo R, Borg RM, Vassallo N, Juge F, Bordonne R, Cauchi RJ. Novel interactors of the Drosophila survival motor neuron (SMN) complex suggest its full conservation. FEBS Lett. 2017;591:3600–14.

    Article  PubMed  CAS  Google Scholar 

  27. Borg R, Cauchi RJ. The Gemin associates of survival motor neuron are required for motor function in Drosophila. PLoS ONE. 2013;8:e83878.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Cauchi RJ, Davies KE, Liu JL. A motor function for the DEAD-box RNA helicase, Gemin3, in Drosophila. PLoS Genet. 2008;4:e1000265.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Fallini C, Bassell GJ, Rossoll W. Spinal muscular atrophy: the role of SMN in axonal mRNA regulation. Brain Res. 2012;1462:81–92.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Cauchi RJ, Sanchez-Pulido L, Liu JL. Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies. Exp Cell Res. 2010;316:2354–64.

    Article  PubMed  CAS  Google Scholar 

  31. Donlin-Asp PG, Fallini C, Campos J, Chou CC, Merritt ME, Phan HC, Bassell GJ, Rossoll W. The survival of motor neuron protein acts as a molecular chaperone for mRNP assembly. Cell Rep. 2017;18:1660–73.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Authors’ contributions

Study design: RJC. Experimentation: BA, RJC. Data analyses: BA, RJC. Manuscript composition and editing: RJC. Both authors read and approved the final manuscript.


The authors thank Matthew Camilleri and Zillah Deussen for technical support. They are also grateful to Marcel van den Heuvel for fly stocks and critical discussions.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Data available on request from the corresponding author.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.


Work on SMN in the authors’ laboratory is supported by the University of Malta Research Fund, and the Malta Council for Science and Technology Internationalisation Partnership Award.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Ruben J. Cauchi.

Additional files

Additional file 1: Table S1.

Chromosome deficiency lines evaluated in the SMN enhancing screen.

Additional file 2: Table S2.

Most frequent Gene Ontology (GO) terms for ‘biological process’ of genes covered by SMN73Ao enhancing chromosome deficiencies. GO terms are ranked in descending order with #1 = most frequent and #14 = least frequent.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aquilina, B., Cauchi, R.J. Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte. BMC Res Notes 11, 378 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: