Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay

Abstract

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

Fig. 1.

Generation of Smg1 gene trap mutant mice. (A) Genomic Smg1 locus (WT) and structure of the RRT449 Smg1 allele containing the pGTOLxf exon trap (Mut). Black boxes, exons; SA, splice acceptor; Beta-geo, β-Geo selection gene; pA, polyadenylation sequence. Arrows indicate forward (Fwd) and reverse (Rev) primer binding sites for PCR genotyping. (B) Single integration of pGTOLxf. Genomic DNA from Smg1^+/+ and Smg1^+/gt ES cells was digested with Xba1 or Sac1 and subjected to Southern blotting to detect pGTOLxf. (C) Embryonic genotyping: genomic DNA from Smg1^+/+, Smg1^+/gt, and Smg1^gt/gt embryos was subjected to PCR to detect the WT Smg1 allele (Fwd/Rev) or the gene trap Smg1 allele (Fwd/GT-rev). (D and E) Confirmation of Smg1 deficiency and reduced Upf1 phosphorylation. (D) Protein lysates from embryonic fibroblasts derived from Smg1^+/+ and Smg1^gt/gt mice were analyzed by Western blotting with anti-Smg1 antibody and with anti–β-tubulin antibody as a recovery and loading control. (E) Immunoprecipitates (IP) collected by using anti-Upf1 antibody from the cell lysates were subject to sequential Western blotting with anti-Upf1 antibody or anti–phospho-S/T-Q antibody to detect phospho-Upf1. β-Tubulin loading control: phospho-specific bands in input (Left) comigrate with Upf1. For all figures, results shown are representative of at least three independent experiments.

Fig. 2.

Morphology and histology of Smg1^gt/gt embryos. Representative age-matched littermate Smg1^+/+ (Left) and Smg1^gt/gt (Right) embryos are shown (N = 48). (A–C) Light microscope images of whole embryos. (D–F) Sequential sagittal sections of E12.5 embryos. Insets: High magnification images (×40). (A) Unturned E8.0 embryos are partially ensheathed by the yolk sac. Extending allantois (A) and somites (S) are visible in both genotypes, but the Smg1^gt/gt embryo lacks the enlarged heart field (H) and indented optic pits (O) visible in the Smg1^+/+ embryo. (B) E10.5 stage. The Smg1^+/+ embryo shows visible delineation of brain ventricles (BV), brachial arches (BA), and typical heart development (H), whereas the developmentally arrested Smg1^gt/gt embryo shows an abnormal heart (H) and pooled blood (Bl) in the thoracoabdominal region. (C) E12.5 stage: Smg1^gt/gt embryo is much smaller than the control and shows inappropriate heart (H) formation and pooled blood (Bl) in the thoracoabdominal region. (D) H&E staining at E12.5 shows the undeveloped tissue architecture of an Smg1^gt/gt embryo. (E) Reduced BrdU incorporation by a Smg1^gt/gt embryo indicates a lack of cell proliferation. (F) Increased TUNEL staining by a Smg1^gt/gt embryo indicates massive cell apoptosis. This embryo is dead and undergoing resorption.

Fig. 3.

Global AS transcript changes in WT versus GT cells measured by RNA-Seq. Total of 2,859 confidently predicted AS events were used to assess changes in splice variant levels (Materials and Methods) between WT and GT cells. (A) Proportion of total events that do or do not contain a PTC for all samples (first column) or for samples that are more than 5% to more than 30% different in %In levels between WT and GT cells (subsequent columns). (B and C) Comparison of AS events grouped in three categories: those introducing a PTC as a result of exon inclusion (PTC upon inc), those introducing a PTC as a result of exon exclusion (PTC upon exc), or those not introducing a PTC (no PTC). (B) Scatter plot showing the distribution of %In levels for WT versus GT cells color-coded according to group (only events with a >5% difference in AS are shown). (C) Mean difference between WT versus GT cells in relative exon inclusion according to group for all samples (first quadrant) and for samples that are more than 5% to more than 25% different in %In between WT and GT (subsequent quadrants). Error bars indicate SEM. (*P < 0.05; **P < 0.005.)

Fig. 4.

AS-coupled NMD regulates the expression of functionally diverse genes. Alternatively spliced variant transcripts of endogenous genes in WT and GT cells were analyzed by RT-PCR. Primers pairs were designed to flank alternative exons that result in PTC introduction by exon inclusion or exon exclusion. Previously validated targets (14) are shown in regular type, and new targets identified by RNA-Seq analysis in the present study appear in bold. Numbers below gels are the mean %In for WT and GT cells as calculated by densitometric analysis of data from three independent experiments.