Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy

Abstract

Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.

Keywords: Abelson interacting protein; Ars2; GARS; PHAX; Prp6; Prp8; RNA-sequencing; SMA; SMN; alternative polyadenylation; alternative splicing; dUTPase; phosphorylated adaptor for RNA export; snRNA; snRNP biogenesis; spinal muscular atrophy; survival motor neuron; αCOP.

FIGURE 1.

Analysis of three different snRNP biogenesis mutants. (A) Browser shots of larval RNA-seq reads at the three disrupted gene loci. Reads from Oregon-R (WT) versus Phax^SH/SH, Smn^X7/D, or Ars2^C/E snRNP biogenesis mutants are shown. Mapped read tracks were normalized to the median of the middle two quartiles of the mapped WT sequence read counts. (B) Northern blot of RNA from snRNP biogenesis mutants and WT larvae. RNA was extracted from WT larvae at 60 ± 2 h (60 h) post egg-laying, and mutant RNA was extracted at 74 ± 2 h to account for their delayed development (see text). (C) Quantification of Northern blot in B. Sm-class snRNAs were normalized to the Lsm-class U6 snRNA, and U6-normalized WT snRNA levels were set at 100. Asterisks are P-values from a Student's t-test: (*) P-value ≤0.05, (**) P-value ≤0.01, (***) P-value ≤0.001.

FIGURE 2.

Gene expression differences in snRNP biogenesis mutants. (A) Heatmap comparison of Cuffdiff FPKM levels of differentially expressed transcripts. Heatmap colors were rescaled for each row and the rows were clustered based on pattern of gene expression between WT and mutants. (B) A heatmap of FPKMs from a set of stress-responsive transcripts in A. (C) Venn diagram of the overlap in gene expression differences in the snRNP biogenesis mutants. Phax = Phax^SH/SH, Smn = Smn^X7/D, and Ars2 = Ars2^C/E. Numbers in parentheses are totals.

FIGURE 3.

mRNA length changes in snRNP biogenesis mutants. Pairwise comparison of significant (false discovery rate adjusted P-value <0.05) differences in mRNA length between WT and Phax^SH/SH (A); WT and Smn^X7/D (B); and WT and Ars2^C/E (C). RNA length differences were extracted from mapped RNA-seq read files by the DaPars linear regression algorithm (see text). Percentage of distal poly(A) site usage index = PDUI. (D) Venn diagram of RNA length differences in snRNP biogenesis mutants labeled as in Figure 2.

FIGURE 4.

Alternative-splicing differences among snRNP biogenesis mutants. Distribution of significant alternative-splicing changes from small |delta Psi| near zero to large |delta Psi| near one: (A) Phax^SH/SH mutants relative to WT; (B) Smn^X7/D mutants relative to WT; and (C) Ars2^C/E mutants relative to WT. (D) Venn diagram of overlapping alternative-splicing changes in snRNP biogenesis mutants, labeled as in Figure 2.

FIGURE 5.

Transgenic rescue of alternative-splicing changes in Phax and Smn mutants. (A) UCSC browser shots of mapped read tracks for four of the top 15 identified alternative splicing changes. (B) qRT-PCR analysis of intron retention in Phax^SH/SH mutants versus Phax^SH/SH mutants expressing a UAS-Phax transgene from the armadillo promoter driven GAL4 (Arm > Phax). (C) qRT-PCR of intron retention in Smn^X7/D mutants versus Smn^X7/X7 mutants expressing a wild-type Smn transgene from its native promoter. For B and C, levels of WT intron retention were set at one. (D) Western blot for dUTPase. Alternate protein isoforms reflect alternative-splicing change in dUTPase mRNA. Wild-type Smn transgene in Smn^X7/X7 background = Tg:Smn^WT. Long dUTPase protein isoform = PA and short = PB.

FIGURE 6.

snRNP-specific protein knockdown in S2 cells. (A) qRT-PCR of untreated cells versus those treated with dsRNA for Smn, Prp6, or Prp8 mRNAs. (B) Western blot of dUTPase levels in RNAi-treated cells. Anti-SMN and anti-α-tubulin verify SMN knockdown and load, respectively. (C) qRT-PCR verification of Prp6 and Prp8 mRNA knockdowns. Levels in untreated cells were set at 100. (D) Northern blot of snRNA levels from S2 cell knockdowns, quantified in E. U6-normalized snRNA levels of WT were set at 100.

FIGURE 7.

Analysis of steady-state snRNA levels and alternative splicing of target genes in SMN missense mutants. (A) qRT-PCR analysis of intron retention of flies expressing SMA patient-derived missense mutations. Transgenic animals of the following generalized genotype were used: Smn^X7/X7, Flag-Smn^Tg/−, where Tg represents a WT, V72G, Y107C, or T205I transgene. Intron retention in the Smn^WT transgenic rescue line was set at one. (B) Northern blots of snRNA levels in the missense mutant lines. (C) Quantification of snRNA levels from panel B. RNA levels in the Smn^WT (WT) transgenic line were set at 100.

FIGURE 8.

Alternative splicing changes in Smn missense mutants. (A) Venn diagram of overlapping alternative-splicing changes in Smn missense mutants. (B) Venn diagram of the overlap in shared splicing differences in the Smn missense mutants with either snRNP-dependent changes or Smn gene-specific changes categorized by comparison of the Phax and Smn total rRNA(−) RNA-seq. (C) Browser shot examples of an Smn gene-specific change in alternative-splicing in scarface (top) or a snRNP-dependent change in CG43394 within a PAPLA1 intron on the opposite strand (bottom). Genotypes are labeled by Smn^Tg, which was expressed in the Smn^X7 null background.

FIGURE 9.

Differential gene expression in Smn hypomorphic animals. (A) Heatmap comparison of Cuffdiff FPKM levels of differentially expressed transcripts. Heatmap colors were rescaled for each row and the rows were clustered based on pattern of gene expression between Smn^WT rescue line and the Smn missense mutants. (B) Venn diagram of overlapping differences in mRNA levels. (C) A heatmap of FPKMs from a set of stress-responsive transcripts. Genotypes are labeled by Smn^Tg, which was expressed in the Smn^X7 null background.