A microRNA feedback loop regulates global microRNA abundance during aging

Abstract

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.

Keywords: Argonaute; Caenorhabditis elegans; aging; miR-71; microRNAs.

FIGURE 1.

alg-1 is an important node in the regulatory gene network during aging. (A) alg-1 and its first neighbors, ordered by highest connectivity (number of unique edges) from 6 o'clock counterclockwise. alg-1 is shown in the center in red. Seventy-two TFs and 26 miRNAs target alg-1. (Yellow nodes) TFs; (blue nodes) aging-associated miRNAs; (pink edges) miRNA-to-gene regulation; (blue edges) TF-to-gene regulation. (B) alg-1 is targeted by 26 different miRNAs through a total of 69 sites. (Left) Edge width reflects the number of target sites for each miRNA in the alg-1 3′ UTR. Nodes ordered by the number of target sites, as in A. (Right) Numeric representation of graphical data on left. (C) 3′ UTR length versus the number of aging-associated miRNA target sites. MiRNA target site numbers retrieved from mirWIP (Hammell et al. 2008). alg-1 is shown by the pink dot. (Red line) Linear fit. (D) Betweenness centrality distribution of nodes. The table (inset) shows the top 10 genes by betweenness centrality index. (Red line) Power-law fit. See also Supplemental Figure S1 and Supplemental Tables S1–S3.

FIGURE 2.

miR-71 directly regulates alg-1 post-transcriptionally. (A) Multiple miRNA target prediction algorithms predict miR-71 target sites in the alg-1 3′ UTR. Black arrowheads indicate miR-71 binding sites that are predicted by >3 algorithms, fall in conserved regions of the nematode genome, and overlap with known ALG-1 binding sites. ALG-1 binding sites (Zisoulis et al. 2010) and PhastCons nematode conservation track (Siepel et al. 2005) retrieved via UCSC Genome Browser (ce6). iCLIP-determined miR-71 binding sites during mid-L4 from Broughton et al. (2016). (B) alg-1 mRNA expression during aging in N2 wild-type background shown as mean ± s.e.m. (n = 2 biological replicates) (normalized to Y45F10D.4 expression). (C–E) alg-1 expression in N2 versus mir-71(n4115) background. (Blue) N2 wild-type or control; (red) mir-71(n4115). (C) alg-1 mRNA expression on Day 3 of adulthood (mean ± s.e.m. [n = 2 biological replicates], normalized to N2 expression levels). (D) Normalized mean GFP reporter expression over whole body at Day 3 of adulthood for control versus mir-71(n4115) background ([*] unpaired t-test, P < 0.01) (mean ± s.d.). (Top) Schematic of GFP reporter construct. miR-71 binding sites indicated by blue boxes. Similarly significantly higher GFP reporter expression was observed in the mir-71(n4115) background on Days 1–9. (E) Protein ALG-1 expression on Days 2 and 5 of adulthood. Protein expression normalized to β-tubulin or actin abundance. (F) (Top) Schematic of 3′ UTR regulation reporter constructs. Control 3′ UTR with wild-type miR-71 binding sites indicated by blue boxes, as in D. Experimental 3′ UTR with mutated miR-71 binding sites indicated by red x. (Bottom) Quantification of reporter expression. Normalized mean GFP signal over whole body at Day 3 of adulthood. Each is average of two integrated strains. GFP reporter expression is significantly higher for mutant 3′ UTR reporter compared with control (unpaired t-test, P < 1 × 10⁻⁹). Mean ± s.d. is shown. Similar results observed for Days 1 and 5 of adulthood. See also Supplemental Figure S2.

FIGURE 3.

mir-71 affects global miRNA abundance. (A) Waterfall plot of miRNA expression fold change during aging in N2 wild-type (blue) versus mir-71(n4115) animals (red). MiRNAs ordered from left to right by magnitude of expression fold change (most positive to most negative) in N2. Corresponding miRNAs are shown in the overlapping position. (B) Histograms of expression fold changes during aging between N2 and mir-71(n4115) animals for microRNAs (top) and mRNAs (bottom). MicroRNA expression shifts globally, with a Spearman's rank correlation coefficient between N2 and mir-71(n4115) of 0.83 (Pearson correlation coefficient = 0.85). mRNA expression does not shift globally but appears to be de-regulated in mir-71(n4115) compared with N2 (Spearman's rank correlation coefficient = 0.55; Pearson correlation coefficient = 0.66). (C) Validation of miRNA expression during aging in N2 versus mir-71(n4115) animals by qRT-PCR. Expression levels were normalized to U18 and respective Day 0/1 time points. (Mean ± s.e.m. n = 2 biological replicates.) Note, y-axis is log scale. See also Supplemental Figure S3 and Supplemental Tables S4–S6.

FIGURE 4.

mRNA expression is dysregulated in aging mir-71(n4115) animals. (A) Comparisons of differentially expressed mRNAs during aging between N2 wild-type (blue) and mir-71(n4115) (red) (P < 0.01). (Left) Venn diagram of up-regulated genes during aging. (Right) Venn diagram of down-regulated genes during aging. (B) Enriched GO terms for differentially expressed genes during aging. Overlay of GO terms for genes up-regulated in N2 (dark blue), genes down-regulated in N2 (green), genes up-regulated in mir-71(n4115) (magenta), and genes down-regulated in mir-71(n4115) (cyan). Y-axis is the log of fractional difference between observed numbers of genes vs. expected numbers of genes. Positive values indicate enrichment and negative values indicate depletion. Statistically significant enrichment of GO term in a given experimental condition indicated by asterisk (*) (hypergeometric test, Benjamini–Hochberg adjusted P < 0.001). Image generated in PANTHER (Mi et al. 2013). (C) Adapted waterfall plot of mRNA expression fold change during aging in N2 wild-type (blue) versus mir-71(n4115) (red). Genes ordered from left to right by magnitude of expression fold change (most positive to most negative) in N2. Corresponding genes are shown in the overlapping position. See also Supplemental Tables S7 and S8.

FIGURE 5.

Gene expression variability during aging and life span variability is diminished in mir-71(n4115) animals. (A) Histogram of noise difference between Day 5 versus Day 0 (Day 5–Day 0) in N2 (left panel) versus mir-71(n4115) (middle panel) (right panel is composite). Y-axis is shown as density rather than counts. Slightly more genes demonstrate positive gene expression noise difference in N2 (+4%, binomial test P = 0.004). More genes demonstrate negative noise difference in mir-71(n4115) (+109%, binomial test, P < 2.2 × 10⁻¹⁶). (B) Histogram of noise difference between mir-71(n4115) and N2 wild-type [mir-71(n4115) − N2] at Day 0 (left panel) versus Day 5 (middle panel) (right panel is composite). Noise was calculated as (σ²)/(µ^1.549275). (C) Life span variability of different strains plotted as CV of life span. Each dot represents a biological replicate experiment. See also Supplemental Figures S4 and S5.

FIGURE 6.

A model for miR-71-mediated regulation of miRNAs. miR-71 is in a composite feedback loop with alg-1. This interaction, by affecting alg-1/Argonaute expression, influences the expression of other miRNAs. Altered miRNA expression seems to in turn affect target mRNA expression and gene expression noise, ultimately affecting longevity.