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, 17 (10), 1804-20

Age-associated Changes in Expression of Small, Noncoding RNAs, Including microRNAs, in C. Elegans

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Age-associated Changes in Expression of Small, Noncoding RNAs, Including microRNAs, in C. Elegans

Masaomi Kato et al. RNA.

Abstract

Small, noncoding RNAs (sncRNAs), including microRNAs (miRNAs), impact diverse biological events through the control of gene expression and genome stability. However, the role of these sncRNAs in aging remains largely unknown. To understand the contribution of sncRNAs to the aging process, we performed small RNA profiling by deep-sequencing over the course of Caenorhabditis elegans (C. elegans) aging. Many small RNAs, including a significant number of miRNAs, change their expression during aging in C. elegans. Further studies of miRNA expression changes under conditions that modify lifespan demonstrate the tight control of their expression during aging. Adult-specific loss of argonaute-like gene-1 (alg-1) activity, which is necessary for miRNA maturation and function, resulted in an abnormal lifespan, suggesting that miRNAs are, indeed, required in adulthood for normal aging. miRNA target prediction algorithms combined with transcriptome data and pathway enrichment analysis revealed likely targets of these age-associated miRNAs with known roles in aging, such as mitochondrial metabolism. Furthermore, a computational analysis of our deep-sequencing data identified additional age-associated sncRNAs, including miRNA star strands, novel miRNA candidates, and endo-siRNA sequences. We also show an increase of specific transfer RNA (tRNA) fragments during aging, which are known to be induced in response to stress in several organisms. This study suggests that sncRNAs including miRNAs contribute to lifespan regulation in C. elegans, and indicates new connections between aging, stress responses, and the small RNA world.

Figures

FIGURE 1.
FIGURE 1.
Lifespan of spe-9(hc88) mutant animals and summary of deep-sequencing reads. (A) Animals were raised and cultured at 23°C in the lifespan assay. Mean adult lifespan was 8.62 +/− 0.05 d. Detailed results of the lifespan assay are shown in Supplemental Table S4. Error bars represent standard error (SE) calculated from triplicates. RNAs were purified at Day 0, Day 5, Day 8, and Day 12 post-L4 molt as highlighted and used for library preparation for Solexa deep-sequencing experiments. (B) Proportion of each noncoding RNA species, including miRNAs, 21U-RNAs/piRNAs, and tRNAs, were analyzed in each aging sample. Details are shown in Supplemental Table S1.
FIGURE 2.
FIGURE 2.
miRNAs with most increased and decreased expression during aging. (A) Of the mature miRNAs with statistically significant expression changes during aging (P ≤ 0.05), those with more than twofold changes in the number of reads from Day 0 to Day 8 are shown here. This list also includes a mature miRNA variant for miR-71 and two miRNA star strands. miRNAs were sorted by the fold-changes. P-values are minimum P-value of pairwise analyses among Day 0, Day 5, Day 8, and Day 12 (see Materials and Methods for more details). Two of the miRNAs with * in their names represent miRNA star species, and sequences with the most abundant number of reads (Supplemental Table S3) were used for counting as we have done for the mature miRNAs. Note that the expression of annotated mature miR-71 (19 nt) appeared to be increased during aging as observed in the qRT-PCR experiments (Supplemental Fig. S2C). However, its longer form (miR-71_L-form) (23 nt) was more abundant than the annotated one, and it showed decreased expression during aging (Supplemental Table S3; details are discussed in the legend for Supplemental Table S2). (B) The results of miRNA expression changes were confirmed by qRT-PCR. The results were normalized by the average of the expression of act-3 and ama-1. Error bars for qRT-PCR results shown by dark gray bars indicate standard deviation (SD). Additional results are shown in Supplemental Figure S2C. Error bars for deep-sequencing results shown by light gray bars represent the maximum and minimum values in fold-changes in two replicates, which were calculated after each read was normalized by the total number of aligned reads in each library.
FIGURE 3.
FIGURE 3.
Lifespan of spe-9(hc88) animals treated with adult-specific RNAi against alg-1. (A) Animals were exposed to RNAi only during adulthood. Mean adult lifespans for the control and alg-1 RNAi were 9.14 +/− 0.33 and 7.89 +/− 0.15 d, respectively (P ≤ 0.0005). Detailed results of the lifespan assay are shown in Supplemental Table S4. Error bars represent standard error (SE) calculated from triplicates. (B) Expression changes of age-associated miRNAs were examined by qRT-PCR. Data were normalized by the average of the expression levels of act-3 and ama-1 genes. Error bars represent SD. Day 0 animals cultured on OP50 bacteria (just before RNAi exposure) were used as a control.
FIGURE 4.
FIGURE 4.
Expression changes of age-associated miRNAs in conditions that modify lifespan. (A) Lifespan of spe-9(hc88) animals was assayed at different temperatures, 15°C, 23°C, and 27°C. Mean adult lifespans were 7.62 +/− 0.15 d, 16.21 +/− 0.37 d, and 6.11 +/− 0.17 d at 23°C, 15°C, and 27°C, respectively (P < 0.0001). Error bars indicate SE. Details are shown in Supplemental Table S4. Total RNAs were purified from Day 0, Day 3, Day 5, Day 8, Day 12, and Day 15 post-L4 molt as highlighted. (B) Expression changes of age-associated miRNAs were examined by qRT-PCR. Data were normalized by the average of the expression levels of act-3 and ama-1 genes. Error bars represent SD. Day 0 animals cultured at 23°C (just before the temperature shift) were used as a control. Additional results are shown in Supplemental Figure S5. (C) Similar to the temperature-shift experiment, a delay or acceleration in expression changes of the age-associated miRNAs was observed in genetically modified lifespan backgrounds. In this study, spe-9(hc88) animals were exposed to each feeding RNAi, including the control empty vector (L4440), from the L1 stage, in order to induce a sufficient RNAi effect.
FIGURE 5.
FIGURE 5.
Age-associated changes in GFP signals expressed from miRNA promoter::gfp transgenes. The vertical axis represents a fold-change in GFP signal intensity, microscopically determined from ∼15–20 individual whole animals at each time point for each line. Error bars represent SE. The blue-colored and green-colored bars represent the GFP signal intensity observed in the transgenic lines cultured at the standard temperature 23°C and the long-lived condition at 15°C, respectively. Scale bars represent 100 μm. Additional results for miRNA::gfp lines were examined, and strain information is shown in Supplemental Figure S6B.
FIGURE 6.
FIGURE 6.
Target prediction of age-associated miRNAs and pathway analysis. (A) Target prediction algorithms combined with gene expression profiles and age-related phenotypes followed by the pathway analysis revealed strong candidate targets of age-associated miRNAs and their possible roles in aging. The expression profiles for protein-coding genes were obtained from Budovskaya et al. (2008), where they used a spe-9(hc88) background and similar conditions for RNA preparation, including the time points during aging. The size of each circle reflects the number of genes identified in each category (but the area of overlapped regions is not to scale completely). (B) Pathway enrichment analysis with KEGG against predicted target genes identified their possible roles in aging processes. The results were sorted by the order of enrichment (number of predicted genes in each pathway). For example, many TCA cycle and related genes were predicted as targets. These serve an important function in mitochondria together with genes classified in oxidative phosphorylation (shown by a broken line). The names of gene pathways shown in black represent genes necessary for the normal lifespan, suggesting that these pathways are involved in aging. Detailed results are available in Supplemental Tables S5 and S6.
FIGURE 7.
FIGURE 7.
Changes in expression of miRNA star strands. (A) Mature miRNAs were mostly more abundant than their star miRNA species (green dots), but in some cases, both mature and star miRNA strands accumulated at similar frequency (black dots), or star strands were much more abundant than mature ones (red dots). (B) Correlation of expression changes between mature and star miRNA strands was tested over the time course of aging (detailed results are shown in Supplemental Table S3). Each dot indicates the miRNAs for which we observed statistically significant age-associated expression changes either in their mature or star strands (mature or star miRNAs with less than 10 reads in total from Day 0 to Day 12 are not included here since their expression change is less reliable due to an extreme low abundance). The results were plotted in ascending order by correlation values from left to right. Of those, blue points represent miRNAs with age-associated changes only in mature strands; red points represent miRNAs with age-associated expression changes only in star strands; green points represent miRNAs with age-associated expression changes in both mature and star strands. As for the five red points, these correspond to miR-229, miR-77, miR-2214, miR-789-2, and miR-788, from left to right. As for the two green points, these correspond to miR-34 and miR-230. (C) Two examples of miRNAs with age-associated expression changes in their star strands showed more abundant reads corresponding to star strands than mature ones (top). The vertical scales on the left represent the number of sequence reads for mature ones, and those on the right are for star strands. Another two examples showed distinct changes in abundance during aging between mature and star strands (bottom). Additional miRNAs, such as miR-54, also appear to have different trends in expression changes between mature and star strands (Supplemental Table S3), although their expression changes were not statistically significant due to a lower number of sequence reads.
FIGURE 8.
FIGURE 8.
Characterization of novel miRNA candidates. (A) The secondary structures of primary miRNA precursors were predicted for novel miRNA candidates using the RNAfold program. Note that mir-5546 (1128878_adh) was also reported in Stoeckius et al. (2009), although it has not yet been submitted to miRBase or WormBase. (B) Some novel miRNA candidates may fall into known miRNA families since they have the same “seed” sequence as known miRNAs. (C) The age-associated expression changes of novel miRNA candidates were confirmed by qRT-PCR. The results were normalized by the average of the expression of act-3 and ama-1. Error bars indicate SD. (D) Levels of novel miRNA candidates were examined by qRT-PCR in mutants of Argonaute family genes [top: alg-1(gk214) and bottom: alg-2(ok304)] at two different developmental stages—the fourth larval (L4) and young adult. The results were normalized by the expression level of U18 and standardized to the level in wild-type N2 in each stage examined. Error bars indicate SD. P-values were calculated by t-test (***: P < 0.0001; **: P < 0.001; *: P < 0.05).
FIGURE 9.
FIGURE 9.
Characterization of additional sncRNA candidates. (A) The total number of unannotated reads starting with a U or a G was examined in each aging stage. 21nt-U-RNAs showed a decrease in expression with age, while 26nt-G-RNAs and their related reads appear to be increased. (B) The total number of 21U-RNA/piRNA reads, including both known and novel ones, was reduced during aging. (C) tRNA-derived fragments were increased in their accumulation during aging, and two examples are shown here. The longest bars represent the annotated mature tRNA sequences, and each shorter bar represents unique sequence reads corresponding to a part of their mature products. The scores and the color gradation on each shorter bar indicate the number of sequencing reads for each read found in all aging samples. The bar graphs represent the expression changes of the total number of tRNA-derived fragments during aging. All examples are shown in Supplemental Table S15.

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