A Drosophila Model of Spinal Muscular Atrophy Uncouples snRNP Biogenesis Functions of Survival Motor Neuron from Locomotion and Viability Defects  Kavita Praveen, Ying Wen, A. Gregory Matera  Cell Reports  Volume 1, Issue 6, Pages 624-631 (June 2012) DOI: 10.1016/j.celrep.2012.05.014 Copyright © 2012 The Authors Terms and Conditions

Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

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 Figure S1. An asterisk marks a heteroallelic variant U11 snRNA present in the Smn null background (see Figure S1). Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

Figure 2 Analysis of Minor-Class Intron Splicing Defects in Smn Null Mutants and U6atac Hypomorphs mRNA levels for 18 genes containing putative U12-dependent introns in ∼76 hr OR, Smn−/−, and U6atac−/− larvae were measured by qRT-PCR and normalized to OR using Rpl32 mRNA. Samples were run in technical triplicates for each of three biological replicates. The error bars indicate standard error of the mean. 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 18 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. Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

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. Approximately 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 coexpressing 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-dSMN. 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-dSMN; antitubulin 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-square test was performed to determine significance. All error bars represent the standard error of the mean. Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

Figure 4 SmnWT and SmnT205I Animals Have Similar snRNA Profiles (A) Northern blot showing levels of major and minor class snRNAs in ∼76 hr 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. Error bars represent the standard error of the mean. See Figure S2 for additional information. Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

Figure S1 Absence of Unassembled snRNAs in Smn Mutants and Predicted Secondary Structure of a U11 snRNA Variant, Related to Figure 1 (A) Total snRNA levels in Drosophila larvae reflect snRNP levels. Lysates from ∼76 hr OR and SmnX7/D larvae were immunoprecipitated (IP) with anti-Sm antibody, Y12. The bound snRNAs were visualized via Northern blotting with probes specific to the indicated snRNAs and 5.8S rRNA, which binds non-specifically to beads and used as an approximate load control. The levels of snRNAs co-precipitated for OR and SmnX7/D were similar to the total level of snRNAs in the input. (B) An illustration of the secondary structures of the canonical and variant forms of U11. The structures are based on those of Schneider et al. (2004) and redrawn using VARNA (Darty et al., 2009). The arrows on the canonical U11 indicate a 22 base stem loop that is deleted in the genomic sequence of the variant allele. (C) Northern blot of RNA from OR, SmnD/X7, SmnX7/X7 and SmnD/D larvae with U11 specific probes. The asterisk marks the variant U11 that migrates as a shorter band in SmnD/X7 and SmnD/D, but is absent in OR and SmnX7/X7. In SmnD/D larvae, which are homozygous for the U11 variant, remnants of the maternally contributed canonical U11 can be seen. (D) The variant U11 is complexed with Sm proteins. Lysates from OR and SmnD/X7 larvae were immunoprecipiated (IP) with anti-Sm antibody, Y12, and RNA was extracted and analyzed by Northern blotting with a U11 probe. Both forms of U11 co-purified with the Y12 antibody. Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions

Figure S2 Quantitative Real-Time PCR Analysis of Minor-Class Introns in Smn Transgenic Rescue Lines, Related to Figure 4 The four genes showing the greatest reduction in Figure 2 and a control gene that was not reduced (CG11984) were analyzed. For CG18177 and CG15081, the values among all three genotypes are not significantly different from each other. For CG11839 and CG33108, values are significant to p ∼0.05 level when comparing OR versus SmnT205I and OR versus SmnWT, respectively. Error bars represent the standard error of the mean. Additional details on quantitation in Figure 4. In Figure 4, all the Sm-class snRNAs, except for U2, were reduced in Smn−/− mutants, with U4, U5, U11, U12 and U4atac (p ∼0.001) showing the greatest reduction. SmnWT and SmnT205I transgenic animals showed increased levels of U12 compared to Smn−/− animals (p < 0.001) but did not show a significant difference in the levels of U1, U4, U11 and U4atac (p > 0.05). SmnWT and SmnT205I animals also displayed a slight increase in U5 snRNA levels compared to the Smn−/− larvae (p ∼0.001 and ∼0.045, respectively), although both remained well below those of OR animals. Cell Reports 2012 1, 624-631DOI: (10.1016/j.celrep.2012.05.014) Copyright © 2012 The Authors Terms and Conditions