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Keywords:

  • aging;
  • longevity;
  • microarray;
  • translational control

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

MicroRNAs (miRNAs) are small, abundant transcripts that can bind partially homologous target messages to inhibit their translation in animal cells. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signaling and oncogenesis. Little is known, however, of miRNA contributions to aging. We examined the expression of 114 identified Caenorhabditis elegans miRNAs during the adult lifespan and find that 34 miRNAs exhibit changes in expression during adulthood (P≤ 0.05), 31 with more than a twofold level change. The majority of age-regulated miRNAs decline in relative abundance as animals grow older. Expression profiles of developmental timing regulators lin-4 and let-7 miRNAs, as well as conserved muscle miRNA miR-1, show regulation during adulthood. We also used bioinformatic approaches to predict miRNA targets encoded in the C. elegans genome and we highlight candidate miRNA-regulated genes among C. elegans genes previously shown to affect longevity, genes encoding insulin-like ligands, and genes preferentially expressed in C. elegans muscle. Our observations identify miRNAs as potential modulators of age-related decline and suggest a general reduction of message-specific translational inhibition during aging, a previously undescribed feature of C. elegans aging. Since many C. elegans age-regulated miRNAs are conserved across species, our observations identify candidate age-regulating miRNAs in both nematodes and humans.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

One striking theme emerging from intensive investigations into the biology of aging over the last decade is that aging and longevity can be modulated by multiple cellular activities, molecular mechanisms of which appear conserved among metazoans (Martin et al., 1996; Partridge & Gems, 2002; Tissenbaum & Guarente, 2002; Tatar et al., 2003). For example, caloric restriction, partial inhibition of insulin-signaling pathways, and high antioxidant defenses extend longevity in nematodes, flies, and mammals, and common molecular machinery is utilized to accomplish this. Even tissue-specific decline, such as sarcopenia (the progressive loss of muscle mass and muscle strength over adult life) (Fisher, 2004; Karakelides & Sreekumaran Nair, 2005), appears a common feature of aging metazoans (Herndon et al., 2002). Changes in gene and protein expression can clearly be factors in conserved aging mechanisms, and genetic engineering in model systems has been shown to modulate lifespan in many independent experiments. Considerable attention has been devoted to microarray screens for protein-encoding genes that change expression in adult life, which as such, might influence aging or longevity (Lund et al., 2002; Weindruch et al., 2002; Murphy et al., 2003; Fraser et al., 2005; Kim et al., 2005). Potential roles of microRNAs in age-related decline, however, have not yet been extensively investigated.

MicroRNAs (miRNAs) are small (∼22 nt), noncoding RNAs that regulate gene expression in multicellular organisms. The first genetically identified miRNAs were Caenorhabditis elegans lin-4 and let-7, which regulate translation of specific transcripts by binding to partially complementary sites in 3′ untranslated regions (UTR) during different developmental stages. The lin-4 and let-7 miRNAs influence timing of developmental events by down-regulating the expression of their targets (Lee et al., 1993; Wightman et al., 1993; Reinhart et al., 2000; Pasquinelli & Ruvkun, 2002; Bagga et al., 2005). It is now appreciated that miRNA modulation of gene expression is a major regulatory mechanism operative in the animal and plant kingdoms. To date, 326 miRNA genes have been annotated in the human genome, 78 in the fruit fly, 114 in the nematode C. elegans and 117 in the plant Arabidopsis thaliana (miRBase: http://www.sanger.ac.uk/Software/Rfam/; Griffiths-Jones, 2004). A significant number of these miRNAs are evolutionarily conserved, with more than one-third of the C. elegans miRNAs having clear homologs among human miRNAs (Lim et al., 2003). miRNAs are expressed at high levels with an estimated average of 1000 molecules per cell and highest levels of up to 50 000 molecules per cell, an abundance that is hundreds, if not thousands, of times greater than that of typical protein-encoding mRNAs (Lim et al., 2003). A given message can be targeted by multiple miRNAs, and conversely, each miRNA may have multiple targets (Bartel & Chen, 2004; Hobert, 2004). As many as 30% of mammalian genes have been estimated to be potential targets of miRNAs (Lewis et al., 2005). Although translational repression is commonly a consequence of miRNA action on a target transcript in animals, mRNA degradation may also result (Yekta et al., 2004; Bagga et al., 2005; Jing et al., 2005; Lim et al., 2005).

The functions of only a few miRNAs have been discerned, revealing that these small regulatory molecules control fundamental biological events such as cell proliferation, cell differentiation, cell death, cell signaling, stress response, and fat metabolism (reviewed in Bartel, 2004; He & Hannon, 2004; Yang et al., 2005). The potential of miRNAs to influence aging has not been widely addressed, although recent studies have elegantly demonstrated how the developmental timing regulator miRNA lin-4 affects C. elegans lifespan (Boehm & Slack, 2005). Here we report a genome-wide analysis of how expression of miRNAs changes during adulthood and aging in C. elegans. We find that roughly half of the nematode miRNAs exhibit changes in expression level during adult life. Included among these are lin-4 and let-7 miRNAs and miR-1, homologs of which are expressed in Drosophila, zebrafish, chick and mammalian muscle (Lee & Ambros, 2001; Mansfield et al., 2004; Wienholds et al., 2005; Zhao et al., 2005) and which can influence embryonic muscle development (Kwon et al., 2005) and maintenance in fly larvae (Sokol & Ambros, 2005). To set the stage for identification of the mRNA targets of age-related miRNAs, we also used prediction programs to identify candidate mRNA transcript targets and highlight several genes that have been implicated in, or are known to affect, aging. Our data identify candidate miRNAs that might modulate sarcopenia, aging and lifespan. Since many age-regulated miRNAs are conserved, we suggest that their human homologs might be similarly regulated to modulate age-related decline.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

MicroRNAs are regulated during adult lifespan

To address whether C. elegans miRNAs are differentially expressed during adult lifespan, we monitored expression of cataloged miRNAs from the first day of adulthood until late into post-reproductive life, when only 7% of the population is still viable (Fig. 1). To avoid contamination of the aging population with young progeny, we used the temperature-sensitive fertility mutant spe-9(hc88), which has been shown in previous work to have a lifespan similar to wild-type N2 nematodes (Fabian & Johnson, 1994). We reared age-synchronized spe-9(hc88) mutants on plates at 25.5 °C (time 0 = egg deposition, day 4 = young adult) and collected ∼250 individuals for small RNA isolation on culture days 6, 8, 11, 13 and 15, performing three independent RNA isolation replicates/hybridizations per time point. We prepared small RNA from 4-day-old animals (3 replicates) for the sample that was used for normalization.

image

Figure 1. Lifespan details of the strain spe-9(hc88) under conditions of small RNA isolation. Sixty animals were scored for viability over adult lifespan at 25.5 °C. Time point 0 corresponds to when eggs are harvested. Graph represents a population of worms used in one of the three repeat trials. spe-9(hc88) is sterile at 25.5 °C due to a defect in spermatogenesis, but lays unfertilized oocytes in early adulthood. The reproductive period was inferred from the period of laying of unfertilized eggs and corresponds to what we observe for the wild-type strain N2.

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We monitored miRNA expression using a DNA oligonucleotide-based array (Goff et al., 2005) that houses tandem dimer probes complementary to mature sequences of known C. elegans miRNAs (miRBase version 5.0). To circumvent potential signal amplification biases introduced by commonly used RT (reverse transcription) and PCR (polymerase chain reaction) amplification approaches, we used the 3DNA Array900 miRNA direct labeling method for quantitation of miRNA abundance. In brief, this method involves tailing the mature miRNAs in the small RNA samples with poly (A), tagging the poly (A)-tailed miRNAs with a capture sequence, and subsequent hybridization to Cy5-labeled 3DNA dendrimers, a protocol with the potential for ∼850-fold signal amplification. We measured hybridization signals for each miRNA and then compared signal strengths as the relative ratio of signal from a given day to that of Cy3-labelled 4-day-old young adults. We used a permutation test to evaluate the statistical significance of relative abundance changes we observed (see Experimental procedures).

Of the 114 C. elegans miRNAs cataloged in miRBase version 5.0, we identified 34 that change in expression during adulthood with a 95% confidence statistic; 50 miRNAs are regulated during adulthood within the 90% confidence level. Figure 2 identifies these miRNAs and indicates their relative abundance levels over adulthood; the probability of their appearing so regulated by random chance is given in Supplementary Table S1. We note that most listed miRNAs (31) vary more than twofold between maximum and minimum hybridization values.

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Figure 2. Fifty Caenorhabditis elegans miRNAs that change in abundance during adult lifespan. Colourgram depicts high (red), average (grey), to low (blue) expression levels and summarizes hierarchical clustering (average linkage correlation) of patterns of miRNAs that change over adult lifespan using the JMP IN 5.1 software (SAS Institute, Cary, NC,USA). D6–D15 represent profiles of average ratio of three independent replicate samples of day 6 to day 15 miRNA divided by day 4 control value. Thirty-four of the 50 shown miRNAs have P-value ≤ 0.05 (indicated by the * symbol), others P-value ≤ 0.1. Refer to Supplementary Table 1 for P-values (permutation trials) and q-values.

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Some miRNAs that share sequence similarities across species have been identified as family members (Ambros et al., 2003; Lim et al., 2003). We noted that in some cases, miRNA members of the same family exhibit similar expression trends over time, suggesting coregulation late into life (expression patterns of identified miRNA family groups are plotted in Supplementary Fig. S1A). miR-10 family members miR-51 and miR-57 show most similar expression patterns among the 50 age-regulated miRNAs and cluster in Fig. 2. The lack of a comprehensive published list of C. elegans miRNA families limited a more exhaustive comparison of expression patterns of miRNA family members.

Some mir genes that are cosituated in chromosomal clusters (mir-42-43 II; mir-64-66 III; mir-250-mir-61 V; mir-54-56 X) exhibit similar temporal expression patterns during adulthood (Supplementary Fig. S1B). miR-64 and miR-65 (in mir-64-66 III cluster), miR-37 and miR-39, and miR-36 and miR-38 (in mir-34-41 II cluster) show most similar expression patterns and are clustered in Fig. 2.

miRNAs that change dramatically in abundance during adult life

Several miRNAs are distinctive in that they exhibit large changes in relative abundance over adult life [see Fig. 3A–C for data on the miRNAs with greatest relative increases (3A, 3B) and decreases (3C)]. In particular, miR-231 (Fig. 3A) is noteworthy for its high level of expression, its net increase in abundance over time (∼8-fold), and its variation over adulthood (increase to day 11, decrease at day 13, strongly increased expression level at day 15 – > 100-fold higher than at day 4). miRNAs that change over adulthood are plausible modulators of gene batteries that could influence the aging process.

image

Figure 3. Distinctive miRNA expression level changes during adult lifespan. (A and B) Five miRNAs that exhibit the greatest fold increase between any two points during adulthood. For this and all graphs in Fig. 3, the X axis corresponds to age as counted with time 0 being point of egg isolation. The Y axis is the average ratio of sample score/4-day-old control score (Cy5/Cy3). Note that miR-231 (A) stands out as the most highly expressed miRNA and changes more than eight-fold in overall abundance over adulthood. This graph features a different Y axis scale than most other figures; other miRNAs with greatest change between 2.9- and 5.9-fold are in (B). (C) Five miRNAs that exhibit the greatest fold decrease between any two points during adulthood. (D) miR-34 increases in abundance in mid-life. Note that miR-34 is the only miRNA that increases in relative abundance during the reproductive phase of life in the group of regulated miRNAs that fall in the 95% confidence level. (E) miRNAs exhibiting significant decreases in abundance in mid-life. Five miRNAs falling within the 95% confidence level decrease in relative abundance between day 6 to day 8. Note that the well-characterized let-7 miRNA is included in this group. (F) miRNAs with peak expression levels at day 15. Five of the 34 miRNAs in the 95% confidence group fall into this category, including miR-231 (3A). (G) miRNAs with lowest expression levels at day 15. Five miRNAs with the greatest fold change at 15 days. Twenty miRNAs exhibit lowest expression at day 15, see Supplemental Fig. S2. (H) Expression of lin-4, let-7 and mir-1 miRNAs over adulthood.

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Are there mid-life crisis miRNAs?

Several studies have shown that critical genetically regulated events can occur early in adulthood to influence aging and lifespan phenotypes expressed later in life (Dillin et al., 2002a,b). Moreover, we have documented that changes in age-associated phenotypes, such as decline in muscle integrity (Herndon et al., 2002) and acceleration of age pigment accumulation rate (Gerstbrein et al., 2005), initiate around the time that animals enter the post-reproductive phase of life. These observations suggest that key gene expression changes that affect aging may occur prior to the end of the C. elegans reproductive phase. We therefore wanted to identify miRNAs that exhibit significant changes in abundance in early adulthood (Fig. 3D,E). The relative levels of six miRNAs change significantly during the reproductive phase of life. miR-34 levels increase substantially between days 6 and 8. Five age-regulated miRNAs, including let-7 miRNA, decrease between days 6 and 8. Importantly, expression of most of these age-regulated miRNAs is similar in wild-type and young glp-4(bn2) mutant adults that lack most germline cells (Lim et al., 2003), indicating that expression changes most likely reflect modulation in the adult soma rather than the lack of developing embryos in the spe-9 mutant. We propose that members of the miRNA group for which relative expression levels change during the reproductive phase might act early to influence aging and lifespan.

A significant change in translational regulation may be induced by changes in miRNA expression

A preponderance of C. elegans miRNAs that change in abundance over adulthood exhibit a downward trend in expression level over time. Since the major effect of miRNA action in animal cells is an inhibition of translation, lowering of miRNA levels would be inferred to induce a general reduction in translational block and a concomitant increase in protein expression of the targeted messages (assuming message transcription/abundance to be constant). Although major changes in abundant protein expression are not characteristic of C. elegans aging (Johnson & McCaffrey, 1985), our data suggest that C. elegans midlife might include a significant shift in specific protein translation that could impact aging biology.

End-stage expression changes

End-stage expression changes might have an impact on, or be diagnostic of, the most advanced stages of age-related decline. Interestingly, despite homogenous genomes and identical culture conditions, individuals within age-synchronized C. elegans populations show considerable variation in how decrepit they appear in older cultures – for example, as judged by locomotory ability and age pigment (lipofuscin) levels (Garigan et al., 2002; Herndon et al., 2002; Gerstbrein et al., 2005). On day 15 of our experiment, only 7% of the cultured animals are alive and most animals are classified into the decrepit end stage of life. Most of the endogenous processes that promote age-related decline have probably already transpired in this population. Transcriptional profiles of miRNAs in such animals might therefore reflect an advanced aging state. From another point of view, however, it can be appreciated that this is the group of animals that survived longest – they might reflect a subset of the population with an optimal stochastically based combination of differential gene expression (for example see Golden & Melov, 2004; Rea et al., 2005) that contributes to longer and healthier life overall. Although it is not possible to evaluate whether the end-stage gene expression changes reflect beneficial or harmful miRNA transcription profiles at this point, we highlight the miRNAs that have highest (five miRNAs; Fig. 3F and miR-231 in Fig. 3A), or lowest (20 miRNAs; Fig. 3G, Supplementary Fig. S2) expression level at day 15. Study of these miRNAs may help resolve how end-stage changes in miRNA expression impact, or report on, the aging process.

Age-modulated miRNAs include timing regulators let-7 and lin-4 miRNAs as well as muscle miRNA miR-1

Developmental timing regulators lin-4 and let-7 miRNAs

Premature expression of lin-4 or let-7 miRNAs causes precocious implementation of specific developmental events, whereas loss-of-function mutations in these well-characterized miRNAs delay these events, establishing a clear role for these miRNAs in developmental timing (Lee et al., 1993; Reinhart et al., 2000). lin-4 is required for transition from the first to the second larval stage, whereas let-7 is essential for viability and for transition from late larval to adult stage. Both lin-4 and let-7 miRNAs are included in the group of miRNAs that are regulated at some point over adult life (Fig. 3H). let-7 miRNA is expressed at highest levels early in adulthood with expression leveling off to minimal values beginning at day 11 and holding at these lower values through the post-reproductive period. lin-4 miRNA also declines to day 11, but exhibits a modest increase in relative abundance at day 13 before returning to levels expressed at day 11. The lin-4 and let-7 expression patterns suggest the potential to influence the relative timing of processes associated with age-related decline. Indeed, lin-4 has been recently shown to be required for normal lifespan through the down-regulation of the transcription factor lin-14 (Boehm & Slack, 2005). Reduced activity of lin-4 shortens lifespan, whereas lin-4 overexpression or reduced activity of lin-14 extends lifespan. Our observation of decreasing levels of the lin-4 miRNA with age fits with lin-4 effects in lifespan, and this example serves as proof-of-principle that age-regulated miRNAs can impact lifespan. It is also interesting to note that validated let-7 targets include the mRNA transcript for nuclear hormone receptor daf-12 (Grosshans et al., 2005). daf-12 loss of function extends lifespan in C. elegans (Hsin & Kenyon, 1999; Boehm & Slack, 2005). The diminishing levels of let-7 miRNA with advancing age and the implication of a known let-7 gene target in lifespan suggest that let-7 could influence lifespan in a similar manner to lin-4.

miR-1

miR-1 is a highly conserved miRNA with common tissue-specific expression in somatic muscle in flies, zebrafish, chick, mice and humans (Lee & Ambros, 2001; Mansfield et al., 2004; Sokol & Ambros, 2005; Wienholds et al., 2005; Zhao et al., 2005). In mammals, expression of miR-1 family members has been implicated in cardiomyocyte and skeletal muscle differentiation (Zhao et al., 2005) and miR-1 overexpression in HeLa cells shifts the transcriptional balance to a profile more akin to muscle transcriptional profiles (Lim et al., 2005). Elegant experimental manipulations in Drosophila have shown that fly miR-1 regulates cell differentiation of specific cardiac cell types during embryonic development (Kwon et al., 2005) and has an essential role in maintaining muscle integrity when larvae enter the rapid growth phase (Sokol & Ambros, 2005). Given that sarcopenia is a phenomenon conserved across species and a logical target for health-span therapeutics, we were particularly interested in how miR-1 abundance changes over adulthood in C. elegans (Fig. 3H). We find that miR-1 concentration exhibits a striking decline during adult life, a pattern that parallels the progressive decline of body-wall muscle integrity during C. elegans aging (Herndon et al., 2002). This expression pattern is consistent with a model in which a drop in miR-1 levels might promote muscle aging in nematodes.

Prediction of mRNA targets for age-related miRNAs reveals candidate transcripts that could impact health- and lifespan

Several algorithms have been published for prediction of miRNA targets (Enright et al., 2003; Lai et al., 2003; Lewis et al., 2003, 2005; Stark et al., 2003; John et al., 2004; Robins et al., 2005) and recent advances in understanding of target constraints have increased prediction power (Brennecke et al., 2005; Robins & Press, 2005). We used a modified scoring algorithm from Robins et al. (2005) to identify candidate miRNA targets among predicted and characterized open reading frames in C. elegans. We considered all C. elegans miRNAs in initial target prediction. As would be expected from previous work indicating that miRNAs tend to have large numbers of targets (John et al., 2004; Grun et al., 2005; Krek et al., 2005; Lewis et al., 2005; Lim et al., 2005), this effort generated an extensive list of candidate targets (Supplementary Table S2). We note that our algorithm predicts the daf-12 nuclear hormone receptor transcript as a target of let-7 miRNA, an interaction that has been experimentally demonstrated and predicted by others (Grosshans et al., 2005; Watanabe et al., 2006). Note that in the general prediction list, other known miRNA/gene target pairs such as let-7/hbl-1 and lsy-6/cog-1 are also predicted. Thus, our target prediction list includes bona fide targets of miRNAs.

Given our interest in how miRNAs might influence the biology of aging, we asked whether genes known to influence health- or lifespan are included within lists of candidate targets. We compiled a list of 204 C. elegans genes known to impact lifespan when mutant, overexpressed, or targeted by RNAi (see Experimental procedures; Supplementary Table S3) and we identified targets with candidate binding sites for the subset of 50 miRNAs (Fig. 2) that change expression levels during adult life (Table 1A). Our algorithm predicted potential gerontogene target transcripts for 31 of the 50 age-regulated miRNAs we highlight. Forty-two of the 204 gerontogenes have 3′ sites that include regions complementary to age-regulated miRNAs. Ten of the gerontogene candidate targets are predicted to be targeted by multiple miRNAs (Table 1B). The candidate miRNA target gerontogenes are involved in diverse cellular processes including gene expression, signaling, energy production, metabolism, cellular structure and stress responses. miRNA targets include regulators of transcription or general protein translation (marked by an ‘M’ or ‘P’, respectively; see Table 1A) and thus changes in miRNA expression may exert a more profound effect on overall gene expression than is reflected by examining individual targets. It is intriguing that many predicted targets correspond to genes currently unknown to impact aging – analysis of these genes might provide new insights into the biology of aging at cell and tissue levels.

Table 1.  Predicted targets of age-regulated miRNAs among identified gerontogenes and insulin genes
(A) Gerontogenes predicted as targets of age-regulated miRNAs. Forty-two genes documented to influence aging and predicted as targets of age-regulated miRNAs are listed, with the number of miRNA complementary sites predicted for each indicated. miRNAs marked with an asterisk have a P-value for regulation (permutation analysis) ≤ 0.05, the remaining unmarked miRNAs have a P-value ≤ 0.1 to > 0.05. Functional information and effects on lifespan are included (see Supplementary Table S3 for more details). Genes involved with, or having a potential role in, transcription (M for mRNA expression) or global translation (P for protein expression) are marked. miRNA targeting of these genes could have a global impact on gene expression. Note that we also identified weak miR-34 and miR-58 sites in the 3′ UTR of the gene let-363, which encodes the TOR ortholog (target of rapamycin) that mediates translational regulation (not shown because score was just below the cut-off value)
miRNANumber of sitesGerontogene target Brief descriptionLifespan
*let-75F11A1.3adaf-12 (M)Nuclear hormone receptorExtended
4F11A1.3b-c   
*miR-11F42A10.4efk-1 (P)Elongation factor-2 kinase/Calmodulin-dependent protein kinase IIIExtended
1F09F7.5 UnknownExtended
1K10B4.3 PH domain protein MeltedExtended
miR-21Y105E8A.7eat-18 (M)Transcriptional activatorExtended
3F46A9.6mec-8 (P)RNA binding protein (contains RRM repeats)Extended
*miR-2291C54G4.6dod-18Predicted nucleic acid-binding protein ASMTLExtended
1ZK524.3lrs-2 (P)Mitochondrial leucyl-tRNA synthetaseExtended
1C53A5.1ril-1 (phi-60)UnknownExtended
1R10H10.2spe-26Kelch family (actin biding) proteinExtended
1Y46H3C.1srw-1007-transmembrane olfactory receptorExtended
*miR-2311F49E12.2dod-23Member of calpain protease familyExtended
1F46A9.6mec-8 (P)RNA binding protein (contains RRM repeats)Extended
*miR-2331T06D8.6cchl-1Holocytochrome c synthase/heme-lyaseExtended
1B0554.6dod-20UnknownExtended
1T27E4.8hsp-16.1Alpha crystallinsExtended
miR-239a1Y54G11A.8ddl-3Kinesin light chainExtended
miR-2412F11A1.3daf-12 (M)Nuclear hormone receptorExtended
2R13H8.1daf-16 (M)Transcription factor of the Forkhead/HNF3 familyShortened
*miR-2511K10C3.6nhr-49 (M)Hepatocyte nuclear factor 4 and similar steroid hormone receptorsShortened
1T10E9.7nuo-2Vigilin; NADH-ubiquinone oxidoreductase, NDUFS3/30 kDa subunitExtended
miR-2572C08H9.5old-1Fibroblast/platelet-derived growth factor receptor and related receptor tyrosine kinasesExtended
miR-2652R13H8.1daf-16 (M)Transcription factor of the Forkhead/HNF3 familyShortened
*miR-2681ZK637.8unc-32Vacuolar H ± ATPase V0 sector, subunit aExtended
1F55B11.3 UnknownExtended
1T05A1.4 Transposon-encoded proteins with TYA, reverse transcriptase, integrase domains in various combinationsExtended
1Y53F4B.23 UnknownExtended
miR-351C10C5.6daf-15 (P)Guanine nucleotide binding protein MIP1Extended
*miR-371C10C5.6daf-15 (P)Guanine nucleotide binding protein MIP1Extended
*miR-381C10C5.6daf-15 (P)Guanine nucleotide binding protein MIP1Extended
*miR-432F46A9.6mec-8 (P)RNA binding protein (contains RRM repeats)Extended
1C09B7.2 UnknownExtended
miR-501F11A1.3daf-12 (M)Nuclear hormone receptorExtended
1R08E3.3 UnknownExtended
*miR-542Y75B8A.33 UnknownExtended
*miR-562Y39H10A.7chk-1Checkpoint kinase and related serine/threonine protein kinasesExtended
miR-571K10B4.3 PH domain protein MeltedExtended
*miR-582M02B7.3osm-3Kinesin-like proteinExtended
*miR-591F11A1.3daf-12 (M)Nuclear hormone receptorExtended
*miR-591F59E12.10ddl-1Predicted coiled-coil proteinExtended
1B0554.6dod-20UnknownExtended
1F09F7.5 UnknownExtended
*miR-631T01B10.4nhr-14 (M)Hepatocyte nuclear factor 4 and similar steroid hormone receptorsExtended
1C10G11.5pnk-1Pantothenate kinase PanK and related proteinsShortened
1F26E4.6 Cytochrome c oxidase, subunit VIIc/COX8Extended
*miR-641C10G11.5pnk-1Pantothenate kinase PanK and related proteinsShortened
1F26E4.6 Cytochrome c oxidase, subunit VIIc/COX8Extended
miR-651F26E4.6 Cytochrome c oxidase, subunit VIIc/COX8Extended
*miR-671C10C5.6daf-15 (M)Guanine nucleotide binding protein MIP1Extended
1T13C5.1daf-9Cytochrome P450 CYP2 subfamilyExtended
1C06E1.10rha-2 (P)DEAH-box RNA helicaseExtended
1F09F7.5 UnknownExtended
*miR-701Y45G12B.1nuo-5NADH-ubiquinone oxidoreductase, NDUFS1/75 kDa subunitExtended
miR-712T06D8.6cchl-1Holocytochrome c synthase/heme-lyaseExtended
*miR-731F02A9.6glp-1Fibrillins and related proteins containing Ca2+-binding EGF-like domainsExtended
1M04D8.1ins-21Insulin-like peptideExtended
1K10B4.3 PH domain protein MeltedExtended
*miR-742C07H6.6clk-2Involved in regualting telomere length and DNA damage responseExtended
1R10H10.2spe-26Kelch family (actin biding) proteinExtended
*miR-851K10D11.1dod-17UnknownExtended
1C08H9.5old-1Fibroblast/platelet-derived growth factor receptor and related receptor tyrosine kinasesExtended
1K08D10.7 Phospholipid scramblaseExtended
(B) Gerontogenes predicted to be targeted by multiple age-regulated miRNAs. See legend of Table 1A for the P-values for regulation of the listed miRNAs
Gerontogene target Brief descriptionLifespanmiRNANumber of sites
T06D8.6cchl-1Holocytochrome c synthase/heme-lyaseExtended*miR-2331
miR-712
F11A1.3daf-12Nuclear hormone receptorExtended*let-75
miR-2412
miR-501
*miR-591
C10C5.6daf-15Guanine nucleotide binding protein MIP1ExtendedmiR-351
*miR-371
*miR-381
*miR-671
R13H8.1daf-16Transcription factor of the Forkhead/HNF3 familyShortenedmiR-2412
miR-2652
B0554.6dod-20UnknownExtended*miR-2331
*miR-591
F46A9.6mec-8RNA binding protein (contains RRM repeats)ExtendedmiR-23
*miR-2311
*miR-432
C08H9.5old-1Fibroblast/platelet-derived growth factor receptor and related receptor tyrosine kinasesExtendedmiR-2572
*miR-851
C10G11.5pnk-1Pantothenate kinase PanK and related proteinsShortened*miR-631
*miR-641
R10H10.2spe-26Kelch family (actin biding) proteinExtended*miR-2291
*miR-741
F26E4.6 Cytochrome c oxidase, subunit VIIc/COX8Extended*miR-631
*miR-641
miR-651
(C) Predicted insulin gene targets of age-regulated miRNAs. See legend of Table 1A for the P-values for regulation of the miRNAs shown
miRNANumber of sitesInsulin gene target 
*let-71C06E2.8ins-9
miR-21F41G3.17ins-15
1C17C3.19ins-12
miR-2411ZK84.7ins-20
*miR-2682ZC334.11ins-27
2F41G3.16ins-14
*miR-581F56F3.6ins-17
*miR-591F52B11.6ins-34
*miR-731C17C3.18ins-13
1M04D8.1ins-21
*miR-742ZC334.11ins-27
miR-811ZK75.1ins-4
miR-821ZK75.1ins-4
*miR-851T10D4.4ins-31
1ZC334.1ins-26
1C17C3.20ins-38
2ZC334.11ins-27

Noteworthy target predictions

The combination of miRNA expression patterns and target predictions holds implications that may underlie new working hypotheses, including:

Insulin signaling may be modulated by age-regulated miRNAs

A conserved insulin-signaling pathway modulates C. elegans lifespan (Tatar et al., 2003). There are 39 predicted C. elegans insulins (Pierce et al., 2001; http://www.wormbase.org), which appear to act through a single insulin-like receptor (DAF-2) (Kenyon et al., 1993). Low levels of DAF-2 receptor signaling inhibit phosphorylation of the downstream DAF-16 FOXO transcription factor, enabling DAF-16 nuclear translocation and transcription of a battery of genes including those that are stress protective (Murphy et al., 2003). Interestingly, miRNA lin-4 appears to impact longevity by acting on target lin-14 to influence the insulin-signaling pathway (Boehm & Slack, 2005).

In this context it is noteworthy that we predict 14 of the 39 insulins to be potential targets of age-regulated miRNAs (Table 1C; 11 of the 50 age-regulated miRNAs listed in Fig. 2 have the potential to target insulins). ins-4 and ins-27 contain 3′ UTR complementary sites for multiple age-regulated miRNAs. Our algorithm predicts that ins-21, knockdown of which extends lifespan (Murphy et al., 2003), may be targeted by miR-73.

It is also worth noting that we identified the daf-16 transcript as a candidate target of the age-regulated miR-241 and miR-265 (Fig. 2). miR-241 expression levels are highest on day 6 and decline on day 8 to remain constant until day 15. In contrast, miR-265 levels show a general increase over adult life, and are high on day 13 and highest on day 15. The reciprocity of the miR-241 and miR-265 expression patterns suggests that DAF-16 protein levels could be regulated by these miRNAs throughout adult life.

Protein synthesis may be globally impacted by miRNAs during aging

Mammalian Raptor (regulatory associated protein of TOR) associates with TOR (target of rapamycin) to form a complex that senses nutrient levels and transduces the signals to the downstream translation machinery (Hara et al., 2002; Kim et al., 2002). C. elegans daf-15 encodes the raptor ortholog, and the lowered gene dosage in daf-15 heterozygotes is associated with lifespan extension (Jia et al., 2004). Our predictions identify age-regulated miR-35, miR-37, miR-38 and miR-67 complementary sites in the daf-15 3′UTR (Table 1A). These miRNAs are expressed at highest levels from days 6–13, and could repress daf-15 mRNA translation over this period. A decrease in DAF-15 and TOR signaling could confer beneficial effects on lifespan. Interestingly, let-363 (TOR) contains weak candidate complementary sites for miR-34 and miR-58 (data not shown as scores are just below our cut-off score for the Table 1A list), so multiple points of translational control might be offered by miRNA regulation.

Age-related muscle decline might include an miRNA-dependent component

Sarcopenia is one of the most prevalent debilitating conditions that affects the elderly (Fisher, 2004; Karakelides & Sreekumaran Nair, 2005). Loss of muscle mass and strength occurs roughly at a rate of 1% per year beginning at midlife, so that by the late 80s or older, individuals face ∼50% reduction in strength. Since sarcopenia appears to be a common feature of metazoan aging (Herndon et al., 2002), we searched a list of genes previously determined to be preferentially expressed in larval muscle (Roy et al., 2002) for potential targets of age-regulated miRNAs. We find that of the 1364 muscle-enriched genes, 232 have potential 3′ UTR binding sites for age-regulated miRNAs (Supplementary Table S4). In particular, 28 muscle-enriched transcripts have sites for the miRNAs that change dramatically in abundance during reproductive adulthood (Fig. 3D,E). These miRNAs might influence the process of muscle decline.

As noted above, miR-1 has been experimentally found to have a role in muscle survival in flies (Sokol & Ambros, 2005). Interestingly, we predict 17 targets in the muscle-enriched list for miR-1, including ifb-2, which encodes an intermediate filament protein. In general, however, the candidate targets included in the muscle-expressed list are not disproportionately concentrated in major muscle-specific proteins, an observation consistent with the finding that for mammalian miR-1, predicted targets are not necessarily in highly expressed muscle-specific genes (Lim et al., 2005). miR-1 might target genes that are not muscle-specific but are critical to muscle maintenance late into life.

A complementary way that our data might be mined to identify potential miRNAs that influence muscle aging is to ask which of the age-regulated miRNAs are most often scored as candidate regulators in the list of muscle-enriched genes. From this perspective it is interesting that miR-268 has 38 predicted targets in the muscle list and miR-85 has 31 predicted targets (miR-1 is predicted to have the third most candidate targets in this list with 17 predicted). We hypothesize that these miRNAs might have greatest impact on muscle aging.

Cautionary notes on interpretation of target predictions

Although interesting potential relationships are suggested by the age-regulated miRNAs we have characterized and the targets our algorithm predicts, we emphasize that the biological action of age-regulated miRNAs remains to be addressed and true targets remain to be experimentally defined. The definition of age-regulated miRNAs in C. elegans and the evaluation of potential targets initiate efforts towards this goal.

Potential of miRNA manipulations in anti-aging therapy

Identification of miRNAs that affect lifespan or the quality of aging (healthspan) would present novel targets for treatment of age-related decline. Given that miRNAs tend to be remarkably conserved across species, further analysis in C. elegans may well highlight strong candidates for miRNAs that could impact human aging.

One obvious option for therapeutic intervention with miRNAs that decline with age is to express one or more from a tissue-appropriate promoter that is active throughout lifespan. miRNAs that increase in abundance during maturity might also exert deleterious consequences on healthspan and/or lifespan. Strategies for in vivo interference with miRNAs include introduction of 2′-O-methyl oligoribonucleotides homologous to miRNAs or to their targets (Hutvagner et al., 2004; Poy et al., 2004), which are fairly resistant to degradation and bind RNA with high avidity. Such oligoribonucleotides can be effective in vivo in C. elegans and mammalian culture cells (Hutvagner et al., 2004), and can be used in conjunction with deletion analysis to test hypotheses regarding potential miRNA roles on health-span and longevity. Should an efficient delivery mechanism be elaborated, such reagents might be used for enhancing health span of higher organisms.

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

We have surveyed expression of miRNAs during C. elegans adulthood to provide a genome-wide description of miRNA regulation as this organism ages. We identify 34 of 114 miRNAs that exhibit significant changes in expression levels during adulthood within a 95% confidence level, suggesting that miRNAs have considerable potential to modulate the biology of aging. Although our data reflect whole organism miRNA levels and cannot reveal tissue-specific changes that may be critical under some circumstances, the definition of age-regulated miRNAs in C. elegans is an important first step in assessing the impact of miRNAs on how an organism ages. Manipulation of miRNA levels by broad and/or tissue-specific overexpression or by genetic deletion will reveal their roles on how well animals age and how long animals live.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

Caenorhabditis elegans strains and culture

Caenorhabditis elegans strains used were N2 Bristol and spe-9(hc88) I (strain BA671) with standard culture protocols (Brenner, 1974) unless otherwise specified. We used the temperature-sensitive fertility mutant spe-9(hc88) (Singson et al., 1998), which produces motile sperm that fail to fertilize, to prevent progeny production in cultures. spe-9(hc88) mutants have been previously shown to have a lifespan similar to wild-type N2 nematodes (Fabian & Johnson, 1994). For miRNA preparation, we initially reared spe-9(hc88) mutants at 15 °C (fertile temperature) and synchronized cultures by preparing eggs from bleached adults. We then reared animals at 25.5 °C on standard NGM plates seeded with OP50 bacteria, transferring adults away from unfertilized oocytes that were laid during the reproductive period at the restrictive temperature. We counted culture initiation as time 0 (egg stage), animals were L1 larvae at day 1, and were in their first day of adulthood at day 3. The transition into post-reproductive life in wild-type and spe-9(hc88) cultures (judged from oocyte laying) occurs between days 6 and 8, so we consider day 8 and later as the post-reproductive phase. For each hybridization sample, we collected 250 aging worms by picking (days 6, 8, 11, 13 and 15) (except one day 13 sample 180; one day 15 sample 228; one day 15 sample 168). We harvested age-synchronized worms from a larger culture for the day 4 sample. We prepared and assayed three independent RNA samples for each time point.

miRNA preparation and hybridization

We extracted low molecular weight (LMW) RNA from nematodes using the mirVana™ miRNA extraction kit (Ambion, Austin, TX, USA) and a Pellet Pestle® Motor (Kentes, Vineland, NJ, USA). We used 200 ng of LMW RNA as input and labeled miRNAs using the Array900 miRNA Direct kit (Genisphere Inc., Hatfield, PA, USA), a tagging method that allows direct labeling of mature miRNAs without PCR amplification, reverse transcription or on-slide enzymatic reactions, bypassing potential artifacts associated with such protocols. The fluorescent signal of each labeled miRNA was amplified approximately 850 times with 3DNA dendromer labeling (compared to conventional end labeling, see Goff et al. 2005). For miRNA expression evaluation, we used a recently developed microarray technology (Goff et al., 2005) that features tandem dimer probes complementary to mature or abridged miRNA sequences of all the identified miRNAs of C. elegans according to the miRBase version 5.0 (http://www.sanger.ac.uk/Software/Rfam/). The array gives significant signal with only 100 ng LMW RNA input (about 1 µg total RNA). We conducted three hybridizations of independently isolated small RNA per time point.

Microarray chips were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA, USA) and median spot intensities were generated using GenePix 4.0 (Axon Instruments). We processed and normalized microarray data using GeneTraffic Duo (Strategene, La Jolla, CA, USA). Data were analyzed by using the JMP IN™ 5.1 software (SAS Institute, Cary, NC, USA). We used the 4-day-old time point for normalization, so that we could compare ratios of a given miRNA relative to the young adult state. We compared expression levels of miRNAs by pairwise analysis between different ages of adult life. To determine the set of miRNAs that have a statistically significant time dependency in their expression profile, we employed a permutation test. We chose this test because it offered the advantages that we did not need to make assumptions about the direction of change and we did not need to assume basic properties of our distribution (normality, equal variances, etc.). We computed the probability that an miRNA expression profile violates the null hypothesis that it is not time dependent. For each miRNA, we summed the variances for all five time points. This value sets a cut-off for the permutation test. Then, for multiple trials, we randomly permutated the expression levels from all 15 measurements (five time points with three independent measurements at each point) and recomputed the sum of variances. The probability that the expression profile is time dependent is the percentage of permutation trials with sum of their variances less than the cut-off. We used 100 000 trials, which was sufficient for all P-values to converge up to four significant digits. Using binomial expansion, and given the expectation of the number of miRNAs with P-values greater and less than a P-value of 0.5 is equal for a random set, we find that 34 microRNAs change expression with time at 95% confidence level. For the 50 microRNAs with P-value less than or equal to 0.1, we expect 45 of these to have real time dependence. Adjusted P-values (q-values) were estimated to measure the proportion of false positives incurred.

Identification of potential mRNA targets

We searched for targets with seed matches of perfect Watson-Crick base-pair complementarity to positions two-eight of the miRNAs (counting from the 5′ end). In order to consider these seed matches as potential target sites, we required a minimal cut-off for binding specificity of the remainder of the miRNA to the target. Recent evidence suggests that this is not required for function in humans, but 3′ binding does occur in studies of C. elegans. We used the scoring algorithm from Robins et al. (2005). The binding cut-off is determined by creating a second-order Markov model of the background for the 3′ UTRs. Running a Monte Carlo simulation of our scoring algorithm on the background, we find a cut-off corresponding to a particular P-value. In our case, we wanted a maximally inclusive threshold; the cut-off was therefore set at a relatively high P-value of 0.1. Target genes were ranked by weighting multiple sites in potential target genes with the scores of each site as in Robins et al. (2005). The target prediction strategy favors false positives over false negatives with the intent of creating an inclusive list.

For focused miRNA target predictions, we compiled a list of 204 C. elegans genes implicated in aging from literature searches. This list includes the genes reported in the three genome-wide RNAi screens (Lee et al., 2003; Hamilton et al., 2005; Hansen et al., 2005) and a list of 39 predicted insulin-like ligands in C. elegans was found at http://www.wormbase.org/primarily derived from Pierce et al. (2001). The list of 1364 genes significantly enriched in the muscle of C. elegans L1 larvae was described by Roy et al. (2002).

miRNA families and genomic clusters

miRNA family members represented in Supplementary Fig. S1A have homologs in vertebrates and/or Drosophila and are from Ambros et al. (2003) and Lim et al. (2003). Note that in Supplementary Fig. S1, the expression patterns of all family members and gene clusters are shown regardless of their age-regulation scores. miRNAs were considered to cluster in the genome if they were situated within ∼ 200-bp distance of one another. miRNA sequences were obtained from miRBase version 5.0 (http://www.sanger.ac.uk/Software/Rfam/).

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank Garth Patterson and members of the Padgett and Driscoll laboratories for comments on the work, and R. Hart and L. Goff for assistance in microarray analysis. This study was supported by grants from the National Institutes of Health to R.W.P. and the National Institute on Aging to M.D. M.D. is a Senior Scholar of the Ellison Medical Foundation.

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  6. Experimental procedures
  7. Acknowledgments
  8. References
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Experimental procedures
  7. Acknowledgments
  8. References
  9. Supporting Information
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