A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects - PubMed (original) (raw)

A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects

Kavita Praveen et al. Cell Rep. 2012.

Abstract

The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects.

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Figures

Figure 1

Characterization of Smn null flies. _A. Smn_-/- mutants begin dying 4 days post egg laying (DPE), with a number of larvae surviving for extended periods without pupariation. Note that by 6 DPE the OR control larvae had all pupated. For each genotype n ≥ 100 larvae, collected on day 1 post egg laying. B. Developmental western blot of dSMN levels in control (OR) and Smn null animals. C. Developmental northern blot showing major and minor class spliceosomal snRNA levels in OR and Smn null larvae. 7SK and U6 snRNA are shown as loading controls. These snRNA levels are reflective of the total snRNP levels, as shown in Fig. S1. An asterisk marks a heteroallelic variant U11 snRNA present in the Smn null background (see Fig. S1).

Figure 2

Analysis of minor-class intron splicing defects in Smn and U6atac mutants. mRNA levels for eighteen genes containing putative U12-dependent introns in ~76 hour OR, _Smn_-/- and _U6atac_-/- larvae were measured by qRT-PCR, and normalized to OR using Rpl32 mRNA. The data represent three biological and technical replicates. Primers were designed to cross the exon junction flanking the U12-type intron, such that only spliced mRNAs would be detected (inset). The _U6atac_-/- larvae showed robust defects in splicing of these introns (avg. = 0.32). Nine of the eighteen mRNAs were reduced in _U6atac_-/- animals with p-values < 0.01, eight had p < 0.05 and the levels of one mRNA approached, but did not reach, significance (CG7892, p ~0.08). In contrast, splicing of these same introns was relatively unaffected in _Smn_-/- animals (avg. = 1.06). Although a majority of the mRNAs were not affected, we note that the four mRNAs showing the greatest decrease in the _Smn_-/- mutants were also the most severely affected mRNAs in _U6atac_-/- mutants. Each of these (CG18177, CG33108, CG15081 and CG11839) had p-values < 0.05, although none were below 0.01.

Figure 3

Transgenic rescue of Smn null animals with an SMA patient-derived point mutation, T205I. A. Analysis of Smn null flies expressing SmnWT and SmnT205I transgenic constructs. ~100 first instar larvae were collected and the fraction of live animals at subsequent developmental stages was measured. B. dSMN(T205I) shows mild oligomerization defects. Lysates from cells co-expressing FLAG and Myc tagged versions of each dSMN point mutant and WT were immunoprecipitated with anti-FLAG antibody followed by western with anti-Myc antibody. C. Western blot of lysates from SmnWT and SmnT205I larvae and pupae probed with anti-SMN. Tubulin was used as loading control. D. Western analysis of lysates from flies heterozygous for the native and Actin5C promoter-driven FLAG-Smn constructs. Blots probed with anti-SMN; anti-tubulin as control. E. SmnWT and SmnT205I animals rescued locomotion defects present in _Smn_-/- larvae. Graph shows the performance of Smn-/-, SmnWT and SmnT205I larvae compared to OR in the righting assay. At least 18 larvae were measured for each genotype and Student's T-test was used to calculate p-values. *, p < 0.05; **, p < 0.01; ***, p < 0.0001. F. Graph illustrating results of the burrowing assay for OR, Smn-/-, SmnWT and SmnT205I larvae. A chi-squared test was performed to determine significance.

Figure 4

SmnWT and SmnT205I animals have similar snRNA profiles. A. Northern blot showing levels of major and minor class snRNAs in ~76 hour larvae from OR, Smn-/-, SmnWT and SmnT205I lines. U6 snRNA was used as a control for loading. B. Graph illustrating the relative levels of snRNAs in Smn-/-, SmnWT and SmnT205I larvae compared to OR. Northern blots from at least four biological replicates were used to quantify snRNA levels, using U6 for normalization. Student's T-test was used for statistical analysis. See Fig. S2 for additional information.

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A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects - PubMed (original) (raw)