The role of the NMD factor UPF3B in olfactory sensory neurons

Abstract

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor (Olfr) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo.

Keywords: NMD; Upf3b; anti-microbial genes; developmental biology; mouse; neuroscience; olfactory receptor; olfactory sensory neuron; scRNA-seq.

Figure 1.. Identification of UPF3B-regulated genes and NMD target genes in the olfactory system.

(A) The weight of Upf3b-null vs. WT (wild type) mice at the indicated time points. Upf3b-null mice gain weight slowly during postnatal development but then reach the weight of WT mice at the last time point (9 weeks), a pattern indicative of a partial olfactory defect. *, p<0.05. (B) qPCR analysis of olfactory marker genes in Upf3b-null and WT OE (n = 6). **, p<0.01; ****, p<0.0001. (C) Heatmap of genes differentially expressed in mOSNs from Upf3b-null (KO) vs. WT mice (four biological replicates from each are shown). Row names labeled as green are Olfr genes. Right, the most statistically significant GO terms associated with upregulated genes (top) and downregulated genes (bottom) after Upf3b loss. (D) A list of most statistically enriched GO terms associated with the 52 high-confidence UPF3B-dependent NMD target mRNAs we identified in mOSNs.

Figure 1—figure supplement 1.. Upf3b-null mice behavior and purified mOSNs.

(A) Behavior analysis of Upf3b-null and control (wild type) mice in response to exposure to animal urine or food. (n = 10). (B) FACS plots of dissociated cells from Upf3b-null and control OEs used to purify Omp-eYPF+ cells for RNA-seq analysis. The y-axis measures YFP expression. For each sample, three male mice (8– to 9-weeks old) were pooled together for sorting. (n = 4).

Figure 1—figure supplement 2.. UPF3B-regulated genes in mOSNs.

(A) Principal component analysis of RNAseq datasets from Upf3b-null (KO) and control (WT) mOSNs. (B) qPCR validation of genes shown by RNA-seq analysis to be upregulated (Adcy6, Fosl2, Gdpd3, and Ptch1) or downregulated (Cw22, Fut10, Olfr827, Olfr855, and Olfr1208) in Upf3b-null mOSNs relative to control mOSNs. The genes were chosen on the basis of having known functions, including in neural development. (n = 3). *p<0.05. (C) Immunofluorescence analysis of OE sections from Upf3b-null and wild-type (WT) samples stained with FUT10 and OMP antibodies. FUT10 was chosen on the basis of having known roles in neural development. Nuclei were labeled with DAPI. (n = 3). (D) Heatmap showing the expression of canonical OSN precursor/OSN markers in the indicated Upf3b-null (KO) and control (WT) mOSN-enriched samples, based on RNA-seq analysis. RNA expression levels are represented on a log10 scale of TPM values plus one (0, not expressed; 4, highly expressed). (E) Heatmap showing the expression of all annotated Olfr genes (in ascending order) in the indicated mOSN-enriched samples. RNA expression levels are represented on a log10 scale of TPM values plus one (0, not expressed; 3, highly expressed).

Figure 2.. The mOSN translome and NMD.

(A) Left, strategy used to define the mOSN translome. Right, RNAseq analysis of the expression of gene markers for HBCs (Krt5), GBCs (Lgr5), and mOSNs (Omp) in the indicated samples. (B) Average inferred translation efficiency (TE) of mOSN mRNAs with the indicated 3’UTR length ranges. *, p<0.05. (C) Average 3’UTR length of mOSN mRNAs with the indicated range of TE values. *, p<0.05. (D) mOSN mRNAs from WT mice stratified by steady-state mRNA level (transcriptome) and TE. The number of genes in each category is indicated. (E) Top enriched GO terms associated with the different categories of genes defined in (D). (F) Analysis of upregulated mRNAs (candidate NMD targets) and downregulated mRNAs (indirect targets) are shown on the left and right, respectively. The average shift in expression in Upf3b-null mOSNs relative to WT mOSNs is shown for mOSNs binned by TE (a and c have the highest and lowest TE values, respectively). *, p<0.05. (G) Scatter plot of the 52 high-confidence mOSN NMD targets, showing TE vs. NMD magnitude (upregulation in Upf3b-null mOSNs). Both values are log2-transformed. (H) Scatterplot showing the TE of mRNAs in Upf3b-null vs. WT mOSNs. (I) mRNAs exhibiting significantly altered TE in response to Upf3b loss. (J) mOSN mRNAs from Upf3b-null (KO) mice stratified by steady-state mRNA level (transcriptome) and TE. The number of cells in each category is indicated.

Figure 3.. Identification of OE cell subsets using scRNAseq analysis.

(A) UMAP plot of OE cells from 4 Upf3b-null (KO) and 4 WT mice analyzed by scRNAseq. (B) Same UMAP plot as is in (A), showing the identity of the different cell clusters. (C) Dotplot depicting the expression of gene markers in the cell clusters defined in (B). (D) Left, UMAP plot of reclustered OSN precursors/OSNs defined in (A). Right, genotype information. (E) Same UMAP plot as in (D), showing the expression of stage-specific markers. (F) Same UMAP plot as in (D), showing inferred cell-cycle phase based on the expression of a large set of G2/M- and S-phase genes (Kowalczyk et al., 2015).

Figure 4.. HBC, GBC, iOSN and mOSN heterogeneity.

(A) Violin plots showing the expression of selective gene markers in the four indicated HBC sub-clusters in WT mice. (B) Monocle trajectory analysis of the HBC and GBC sub-clusters we identified. The arrow indicates the inferred direction of differentiation. (C–E) Violin plots showing the expression of selective gene markers in the indicated GBC, iOSN, and mOSN sub-clusters in WT mice. (F) The most statistically enriched signaling pathways in the mOSN-1,–2, and −3 sub-clusters.

Figure 5.. Gene groups exhibiting distinct expression dynamics during OSN development.

(A) Monocle pseudotime trajectory analysis of the indicated cell clusters and sub-clusters from WT mice defined in Figure 3B (top) and Figure 3D (bottom), respectively. (B) Heatmap depicting the expression pattern of the four gene groups we defined, each with a unique expression pattern, as defined by the trajectory timeline shown in (A), upper. Top: pseudotime directions; right: the number of differentially expressed genes in each group and representative biological processes and P-values. (C) The most statistically enriched signaling pathways corresponding to each of the four gene groups defined in (B). (D) Dot plot showing genes related to the Hippo signaling pathway are primarily expressed in HBCs. (E) Transcription factor genes exhibiting the most statistically enriched expression in each gene group defined in (B). Target sequences predicted by the ENCODE database are indicated.

Figure 6.. UPF3B shapes olfactory neurogenesis.

(A) The fraction of HBCs per all OSN precursors/OSNs (HBCs, GBCs, iOSNs and mOSNs) (left) or all OE cells (right), in Upf3b-null (KO) and WT mice, as determined by scRNA-seq analysis. *, p<0.05. (B) The percentage of cells from the indicated cell sub-clusters in Upf3b-null (KO) and WT mice, as determined by scRNAseq analysis. (C) Cell number in each cell sub-cluster, as defined in Figure 3D. (D) Most statistically enriched GO terms in the mOSN-2 and −4 sub-clusters. (E) Heatmap depicting the expression pattern of anti-microbial genes in the indicated cell subsets. (F) Left: Western blot analysis of endogenous CAMP protein level in the OE from Upf3b-null (KO) and WT mice. Right: quantification of CAMP level normalized against GAPDH (n = 3). *, p<0.05. (G) IF analysis of adult mouse OE sections co-stained with antisera against CAMP (red) and OMP (green). Nuclei were stained with DAPI (blue). (H, I) The percentage of mOSNs (H) and iOSNs (I) in our scRNAseq datasets that express Olfr genes. Left, all known Olfr genes. Right, the 78 Olfr genes significantly downregulated in Upf3b-null mice, based on RNAseq analysis (Figure 1C). *, p<0.05.

Figure 6—figure supplement 1.. Impact of UPF3B loss on OE cell subsets.

(A) Left, IF analysis of adult mouse OE sections co-stained with antisera against TRP63 (red) and OMP (green). White arrows label TRP63+ HBCs. (n = 3). Right, quantification of TRP63+ cells per 1 mM. For each group, 15 different OSN field lengths (75 μm long) from three individual mice were examined to calculate the number of TRP63+ cells per mM. (B) The fraction of different cell subsets per OSN precursors and OSNs (HBCs, GBCs, iOSNs and mOSNs) cells (top) or all OE cells (bottom), in indivdiual Upf3b-null (KO) and control (WT) mice, as determined by scRNA-seq analysis. (C) Left, western blot analysis of OMP protein expression in independent OE samples from Upf3b-null and control (wild type) mice. GAPDH is the normalization control. Right, quantification of OMP expression normalized to GAPH levels (n = 4).

Figure 6—figure supplement 2.. mOSN subsets and UPF3B-regulated genes.

(A) Scatter plots showing Pearson correlation analysis of the transcriptomes from the indicated mOSN cell sub-clusters. (B) Left, IF analysis of adult mouse OE sections co-stained with antisera against CAMP/CRAMP (red) and OMP (green). Nuclei were stained with DAPI (blue). (n = 3). Right, quantification of OMP+ cells per mm². For each group, 6 different mSONs views (2500 μm²) from 3 individual mice were quantified to calculate cell number per mm². (C) The chromosomal distribution of the 78 Upf3b-regulated Olfr genes.

Author response image 1.. IF analysis of adult mouse OE sections co-stained with antisera against CAMP (red) and OMP (green).

Nuclei were stained with DAPI (blue).

Author response image 2.. Venn diagram showing the expression of Olfr genes in Upf3b-null and control mOSNs.