Fig 2: Two-factor authentication model of NMD.a Overview of postulated two-factor authentication model. To grant access to the degradative activities of NMD, at least two consecutive security checks need to be passed. Aberrant translation termination in combination with an enhanced residence time of UPF1 on the mRNA and NMD-activating features (e.g., the presence of a downstream EJC) lead to increasing UPF1 phosphorylation by SMG1 (1st authentication step). Sufficiently phosphorylated UPF1 (p-UPF1) leads to the recruitment of SMG5-SMG7 and consequently enables the endonucleolytic activity of SMG6 (2nd authentication step). Domains and functions of SMG5 and SMG7 required to support NMD are indicated (green = low importance, orange = medium, red = high). b Hypothesis for the potential mechanism of the second authentication step. According to this hypothesis, SMG6 can be bound to p-UPF1 before SMG5-SMG7. The role of SMG5-SMG7 in NMD would be the activation of p-UPF1-bound SMG6.

Fig 3: DHX34 can bind SMG1 via its CTD domain or via binding to UPF1.(a) Overlap between the structures of SMG1C–DHX34 and SMG1C-UPF1 (EMD-2264)21, revealing that the formation of a putative SMG1C-UPF1-DHX34 complex is structurally feasible. The putative location of the SMG1 kinase domain is indicated as PIKK in red. Scale bar, 5 nm. (b) Cartoon depicting the domain structure of DHX34 full-length (FL) and C-terminal deletion mutants. (c) HEK293T cells were transfected with full-length T7–DHX34 and DHX34 C-terminal deletion mutants shown in b. Inputs (0.5%) and anti-T7 immunoprecipitates (20%) were probed for the presence of SMG1. Expression of T7–DHX34 constructs was detected with an anti-DHX34 antibody. (d) Analysis of the binding of T7–DHX34 (FL and C-terminal deletion mutants) to FLAG-UPF1. HEK293T cells were transfected with full-length T7–DHX34 or DHX34 C-terminal deletion mutants and FLAG-UPF1. Inputs (0.5%) and anti-FLAG-immunoprecipitates (20%) were probed for the presence of the T7–DHX34 constructs. (e) Sequential co-IPs reveal a SMG1, UPF1 and DHX34 multi-protein complex. Cells expressing FLAG-SMG1, MYC-UPF1 and T7–DHX34 were subjected to anti-FLAG-IPs. FLAG-immunoprecipitates were then eluted using a FLAG peptide and immunoprecipitated using anti-T7 agarose. Inputs (0.5%) and anti-FLAG-IPs (20%) and anti-T7-IPs (10%) were probed with the indicated antibodies. (f) Purification of a tripartite containing SMG1, UPF1 and DHX34 as in e but with IP of FLAG-UPF1 (wild-type and C126S mutant), followed by IP of T7–DHX34 using anti-T7 agarose. Inputs (0.5%), anti-FLAG elutions (10%) and anti-T7-IPs (20%) were probed with the indicated antibodies.

Fig 4: Characterization of the transcriptome of upf1 mutants at 5 dpf. (A) Differential transcript-level expression analysis of wild-type and mutant fish (Datasets S2 and S3). Expression levels are depicted as TPM. (B) Enlarged representation of data in A, focusing on genes with low expression levels. The two most highly expressed NMD transcripts (rpl22l1 and rpl10a) are indicated by green circles. (C) Increased NMD ratios in upf1 mutant fish; four selected genes are highlighted by green circles. For every gene, TPM counts were separately summed for NMD and non-NMD transcripts; the ratio was determined as division of NMD counts by the sum of NMD and non-NMD counts. (D) Violin plot of NMD ratios (P = 4.37 × 10-11). (E) GO term analysis of up-regulated non-NMD transcripts, indicating the enrichment of major categories. (F) GO term analysis of up-regulated NMD transcripts.

Fig 5: Altered landscape of pre-mRNA splicing in upf1 mutants. (A) Examples of genes encoding splice activators (srsf) and repressors (hnrnp), the transcripts of which are dysregulated in upf1 mutants as determined by transcript-level RNA-seq quantitation. (B) Summary of alternative splicing analysis. (C) Stratification of alternative splicing events. (D) Sashimi plots for three different genes indicating three different types of alternative splicing events; the affected exons are boxed. The NMD-transcript identifiers are as follows: srsf7a, ENSDART00000147803.3 (alternative donor site usage leading to inclusion of parts of intron sequences); srsf5a, ENSDART00000149400.2 (exon inclusion); hnrnph3, ENSDART00000108951.4 (exon exclusion/skipping). (E) Quantification of alternative splicing events resulting in generation of a PTC in the affected transcripts of srsf7a (inclusion of intron sequences), srsf5a (inclusion of exon), and hnrnph3 (exon inclusion equivalent to lower inclusion values) in upf1 mutant fish. (F) Higher levels of NMD transcripts of srsf7a, srsf5a, and hnrnph3 as a result of changes in alternative splicing events increase NMD/non-NMD transcript ratios. Black circles, upf1+/+; red circles, upf1-/-.