Dampening of expression oscillations by synchronous regulation of a microRNA and its target

Abstract

The complexity of multicellular organisms requires precise spatiotemporal regulation of gene expression during development. We find that in the nematode Caenorhabditis elegans approximately 2,000 transcripts undergo expression oscillations synchronized with larval transitions while thousands of genes are expressed in temporal gradients, similar to known timing regulators. By counting transcripts in individual worms, we show that pulsatile expression of the microRNA (miRNA) lin-4 maintains the temporal gradient of its target lin-14 by dampening its expression oscillations. Our results demonstrate that this insulation is optimal when pulsatile expression of the miRNA and its target is synchronous. We propose that such a miRNA-mediated incoherent feed-forward loop is a potent filter that prevents the propagation of potentially deleterious fluctuations in gene expression during the development of an organism.

Figure 1. A number of genes display cycling expression dynamics in synchrony with the molting cycle during larval development

(a) Experimental set-up. After synchronizing hatched worms by starvation, samples were collected every 2 hours (1.5 hours) during larval development at 20°C (25°C). For each sample, transcript expression was measured by poly(A)⁺ RNA-seq. (b-c) Normalized expression profiles of all 231 genes (grey) that co-cluster with the collagen encoding molting gene dpy-10 (red) known to exhibit periodic expression. Cycling expression was observed at 20°C (b) and 25°C (c). The average profile is indicated by the broken black line. All curves are spline fits of the discrete expression profiles (red dots for dpy-10). Dashed lines separate larval stages.

Figure 2. Clusters of genes with cycling or graded expression show differential signature of post-transcriptional regulation

(a) Average normalized expression profiles of distinct expression clusters (Supplementary Table 1) for cycling genes (left) and graded genes (right) at 20°C (blue) and 25°C (red). The transparent shading indicates the standard deviation. For developmental times larger than 38 hours at 20°C, data from an independently grown worm population were used. Developmental time at 25°C was rescaled linearly by a factor of 0.77 to account for faster development and make expression at different temperatures comparable (Supplementary Fig. 2). Broken grey lines indicate transitions between larval stages at 20°C. (b) Upper panel: Enrichment of human orthologs (blue) as a proxy for gene age, of lethal RNAi phenotypes (brown) to measure the fraction of genes essential for survival, and of predicted conserved microRNA targets to estimate the extent of post-transcriptional regulation for each cluster. Error bars are based on random counting statistics. P-values were computed by Fisher's exact test. Data are shown for all clusters and for the ensemble of all clustered genes. Lower panel: Percentage of conserved nucleotides within aligned contigs of total exonic sequence (blue), of 3’UTR exons (orange) and for 1 kilobase of upstream sequence (brown). Conservation was measured using multiple sequence alignments of C. elegans, C. briggsae and C. remanei, downloaded from the UCSC genome browser. Unaligned sequence of C. elegans was counted as mismatches to obtain a conservative estimate. Shown are the interquartile range (box) and the median (black). Data are shown for all clusters and for the ensemble of all clustered genes. The colored lines indicate the median across all genes. P-values were computed by a two-sided t-test. (*P<0.05; **P<0.001).

Figure 3. Predicted targets of many microRNAs are enriched in temporally co-expressed genes

(a) Fold enrichment of predicted microRNA targets in cycling expression clusters. Targets of miR-230 are strongly over-represented in cycling clusters with pronounced amplitudes, while miR-1/796 targets are enriched in cycling clusters with low amplitudes. In contrast, predicted miR-71 targets are enriched in all cycling clusters. (b) Fold enrichment of predicted targets of developmental microRNAs. Targets are either dispersed across different expression clusters (miR-51 and let-7 family) or concentrated in a single expression cluster (lin-4 family). Error bars in a and b are based on random counting statistics. P-values in a and b were computed by Fisher's exact test. (*P<0.05; **P<0.001).

Figure 4. lin-4 microRNA expression is pulsatile

(a-b) Mean mature lin-4 level of staged larvae determined by Taqman qPCR and 20°C (a) and 25°C (b). Error bars represent standard deviation (n>3). Dashed lines separate larval stages. (c) Plin-4::gfp smFISH (green) and DAPI (blue) images of staged larvae are merged and ordered by their body lengths. Scale bar indicates 100 μm.

Figure 5. lin-14 mRNA is homogeneously expressed in somatic tissue of worm larvae and exhibits a temporal gradient

(a) Maximum z-projection of lin-14 smFISH stack images of staged larvae. Transcripts are visualized as countable fluorescent spots (inset at 8X zoom). Yellow dashed lines outline the germ line and gonad. Scale bars indicate 100 μm and 12.5 μm (inset) respectively. (b) Body length determined from DAPI staining. (c) Overall lin-14 mRNA concentration of individual animal as a function of body length. The curve indicates a moving average (bin; 36 μm). Dashed lines separate larval stages.

Figure 6. lin-14 mRNA level exhibits pulsatile dynamics in the absence of lin-4 negative regulation

(a-b) lin-14 mRNA concentration dynamics of individual lin-4(e912) mutants as a function of body length. Shown are the overall concentration (a) and the local concentration in hyp8-11 (b). Curves in a and b indicate moving averages (bin size = 36 μm) and dashed lines separate larval stages. (c) AP map of the lin-14 mRNA level fold repression in wild-types compared to lin-4(e912) mutants. AP (%) indicates relative position along AP axis (0%-head, 100%-tail). The height of each bin is 1% and a moving average (bin size = 36 μm) is applied in x-direction. (d) lin-14 mRNA fold repression in the mid-body. Each curve represents trajectory along x-axis of the heat map in c where its corresponding colorbar is located.

Figure 7. Synchronous expression of microRNA and its target dampens target gene expression fluctuations

(a) Mean mature levels of lin-4 (from Fig. 4A, normalized to U18) in animals with wild-type background (red) and lin-14 mRNA concentration in lin-4(e912) mutants (black) oscillate in phase. Expression was normalized by the mean in order to plot data on the same scale. Body length was converted to developmental time using the regression in Supplementary Figure 3a for lin-4(e912) animals. Developmental time was rescaled for the lin-14 profile to account for slower development of lin-4(e912) animals (Supplementary Fig. 13a). Data for lin-14 were binned (bin size = 10 μm) before conversion and error bars represent variability across five independent replicates. (b) A biochemical model of miR-IFFL to describe target and microRNA level dynamics. The target mRNA and microRNA are coherently produced, and decay at constant rates ɣ_R and ɣ_m, respectively. microRNA-mediated mRNA degradation occurs with rate k_on,, which is a function of the number of LCE (N), and microRNA is recycled. (c) k_on fit values for lin-14 alleles with different N. Error bars correspond to 95% confidence interval. (d) miR-IFFL performance landscape. Damping is efficient when microRNA is expressed synchronously to its target (lin-4 microRNA relative fluctuation > 0) with an optimal value of k_on (dark blue valley). Asterisks indicate lin-14 alleles with different N.