HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B896-B901 (2004)
© 2004 The Gerontological Society of America

Age-Related RNA Decline in Adult Drosophila melanogaster

Nuzha M. A. Tahoe, Ali Mokhtarzadeh and James W. Curtsinger

Department of Ecology, Evolution and Behavior, University of Minnesota, St. Paul.

Address correspondence to Nuzha M. A. Tahoe, PhD, Department of Ecology, Evolution and Behavior, University of Minnesota, 100 Ecology, 1987 Upper Buford Circle, St. Paul, MN 55108. E-mail: nmtahoe{at}tc.umn.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
We investigated the correlation between age and total RNA levels in long-lived and control lines of Drosophila melanogaster. Total RNA was extracted at 10 ages from 1–63 days posteclosion from 3 inbred lines, with replication. Three different methods of RNA quantitation gave highly correlated estimates. Total RNA declined substantially with age, exhibiting a dramatic drop in the first few days of adult life. We find no evidence for a causal relationship between adult longevity and total RNA levels, since long-lived and control lines exhibited similar patterns of age-related RNA decline. These observations suggest that the dramatic decline in total RNA that occurs early in adult life does not explain the twofold differences in life span between lines. The pattern of age-specific decline coincides with published observations on age-specific metabolic rates, and suggests that 14-day-old flies are functionally senescent.

Age-related variations in total nucleic acid levels have been studied in Drosophila melanogaster and several other dipteran species, in some cases with contradictory results. There have been reports of increasing nucleic acid content during the first 5 days of adult life in D. melanogaster and the mosquito Aedes aegypti (1,2), while constant levels throughout adult life were reported for the housefly Musca domestica (3). Other studies report age-related declines in nucleic acid content in various animals, including D. melanogaster (4–9). Total RNA, tRNA, rRNA, and many specific mRNAs decrease with increasing adult age in D. bipectinata (9) and D. melanogaster (10–13). Also, RNA content per egg shows a small decrease with maternal age in D. melanogaster (14). Other studies have demonstrated that several mitochondrial transcripts decline in aging flies (15,16). Brack and colleagues (13) suggest a causal connection between total RNA levels and life span, reporting that the decline in total RNA can be slowed by feeding D. melanogaster with the antioxidant N-acetylcysteine, subsequently increasing adult survival.

Among the Drosophila studies that report age-related decline in total RNA levels, there is good agreement on timing. There is a drastic decrease in total RNA at earlier ages of adult life, and then a slower decline at later ages, eventually reaching ~60% in old flies compared with young flies (11–13). A similar decline in protein levels has been reported (11), possibly due to down-regulation of RNA polymerase-mediated transcription. In many animal species as well as in cultured cells, age-related changes in the rate of protein synthesis have been observed (17,18).

Two alternative hypotheses concerning the relationship between life span and RNA levels have been postulated by Shikama and Brack (11): Either the drastic decline in total RNA causes senescence and age-related decline in protein synthesis that is detected in aging flies, or it is unrelated to aging in adults, reflecting the transition from larval to adult developmental stages. In the latter case, it is proposed that enhanced gene expression is necessary for a holometabolous insect to undergo transformation from larvae to adult, but subsequently the nucleic acids are largely nonfunctional and decline with age. In contrast, the former alternative proposes a causal connection between RNA levels and senescence. Shikama and Brack (11) suggested that these hypotheses could be distinguished by comparing RNA levels in lines of Drosophila that differ greatly in life span, but did not pursue that line of experimentation.

Here we study age-specific RNA levels in adult D. melanogaster from genetically homogeneous lines that differ dramatically in patterns of senescence (19). We use recombinant inbred (RI) lines derived from Luckinbill's (20,21) artificial selection experiment for increased life span, which live about twice as long as unselected controls. The long-lived RI lines have normal resting metabolic rates (22), and normal levels of xanthine-related antioxidant enzymes (23). We use males exclusively, because female mass and RNA content can vary substantially depending on oviposition patterns. Because of the well-known sensitivity of RNA quantitation to details of experimental method, we employed three different quantitative methods. We ask how total RNA levels change with adult age, and also whether long-lived and control lines differ with respect to age-specific RNA patterns.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Fly Stocks
We used males from three inbred lines of D. melanogaster: RI7 and RI23 are long-lived recombinant inbred (RI) lines, and 1S9 is an unselected inbred control. All three lines derive from the same genetically heterogeneous wild stocks; for details of stock construction see Curtsinger and Khazaeli (19). Male flies from recombinant inbred lines RI23 and RI7 exhibit average adult life spans in the range 60–80 days, while males from the unselected control line 1S9 live 35–45 days on average (19). Stocks are maintained on standard agar-yeast-molasses-cornmeal medium and are kept at 24°C, with constant light and 65%–70% relative humidity in a walk-in incubator. Approximately 600 male flies were collected at emergence from each line, maintained in population cages, and then sampled for RNA assays at ages 1, 3, 5, 13, 20, 33, 45, 55, 60, and 63 days posteclosion.

RNA Preparation
Total RNA was extracted from groups of 30 male flies of the same age and genotype by freezing under liquid nitrogen and then immediately homogenizing in 800 µl TRizol reagent (Gibco, Carlsbad, CA). RNA was purified following TRizol protocol. Purified RNA was precipitated using isopropyl alcohol and washed twice with 75% ethanol. Some RNA samples (see below) were treated with DNase I RNase free (Ambion, Inc., Austin, TX) and incubated at 37°C for 10 minutes. Excess units were used to ensure complete degradation of DNA contaminants.

Measuring RNA Concentrations
Our primary method for RNA quantitation is ultraviolet (UV) spectrophotometric analysis using GeneQuant RNA/DNA Calculator (Pharmacia LKB Biochrom Ltd., Cambridge, England). For each age and genotype, two independent samples (diluted 40-fold) were assayed by the UV method. In addition, for half of the homogenates (one sample for each age and genotype), we also estimated RNA concentrations by two other methods: RiboGreen Quantitation (High Range Assay) (Molecular Probes, Eugene, OR), and the Agilent 2100 Bioanalyzer Nano assay (Agilent Technologies, Palo Alto, CA). The use of three different quantitative methods is helpful for verifying RNA estimates and identifying aberrant readings.

For the RiboGreen method, we used a Bio-Tek Fluorometer FL600 fluorescence microplate reader (Bio-Tek, Winooski, VT) in combination with the RiboGreen RNA quantitation kit, RiboGreen reagent, and Ribosomal RNA standard (Molecular Probes). RNA samples (200 µl) were pipeted into microplate wells and fluorescence measurements were made using the Microplate Reader (Bio-Tek Instruments, Inc.) with excitation and emission set at 485/20 and 530/20, with shaking and temperature control off. KC4 software (Bio-Tek) was used. A standard curve was generated for each plate from known ribosomal RNA standard concentrations. Both pre- and post-DNase treated RNA preparations were assayed using the RiboGreen method.

For the Agilent method, RNA integrity and quantity was assayed using the Agilent 2100 Bioanalyzer, in combination with a RNA 6000 Nano Lab Chip kit (Agilent Technologies). RNA samples were prepared to a final concentration of 200 ng/µl, based on concentrations determined using the UV method. All samples and the ladder were heat-denatured at 70°C for 2 minutes. Prior to using the Agilent 2100 Bioanalyzer, the electrodes were decontaminated using the electrode cleaner chip and RNaseZAP (Ambion). Other procedures, including preparation and loading of gel-dye mix (9 µl), RNA 6000 Nano marker (5 µl), RNA samples (1 µl), and RNA 6000 ladder (1 µl) in the appropriate LabChip wells, were as instructed by the Agilent manual. All unused sample wells were loaded with 6 µl Nano marker. The chip was vortexed to avoid air bubbles and run in the Agilent 2100 Bioanalyzer within 5 minutes using the Eukaryote Total RNA Nano assay. Default parameters were used as recommended by the Agilent software.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Age-specific measurements of total RNA µg/ml of 30-fly homogenate observed in three inbred lines are shown in Table 1. There are no observations for flies older than 45 days for line 1S9 or 55 days for line RI23 because insufficient flies survived to those advanced ages. UV entries are the means of two independent samples for each age and genotype; other entries in Table 1 are single observations. UV, RiboGreen, and Agilent methods produced highly correlated estimates of age-specific RNA concentrations, with all pair-wise correlation coefficients greater than 0.90 (p <.001 in all cases). For all lines and all measurement techniques, we observed a dramatic decline in total RNA at the earliest ages. Total RNA concentration on Day 5 were 40%–50% of concentrations on Day 1 (Table 1, Figure 1). On Day 13, in all cases, total RNA was less than 40% of concentrations on Day 1. We find no evidence for statistically significant decline after Day 20.


View this table:
[in this window]
[in a new window]
  		Table 1. Quantitative Comparison of Total RNA Concentrations in µg/ml per 30 Adult Male Drosophila Melanogaster at Different Ages 1–63 Days Posteclosion.

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. RNA concentrations (µg/ml per 30 adult male Drosophila melanogaster) assayed by three methods in three inbred lines (1S9, RI7, and RI23) at 10 ages from 1–63 days posteclosion. A, ultraviolet spectrophotometer; B, RiboGreen assay with no DNase treatment; C, RiboGreen assay with DNase; D, Agilent 2100 bioanalyzer. Estimates from the three methods are highly correlated. For all lines and measurement techniques there is a dramatic decline in total RNA observed early in adult life

 
DNase treatment is used to eliminate DNA contamination in RNA preparations. We investigated the possibility of DNA contamination by assaying homogenates before and after DNase treatment. Only one method, RiboGreen, was used for this set of assays, because the three methods are highly correlated. The data, shown in Table 1, indicate that DNase treatment reduces estimated RNA concentrations by ~ 20%, but does not alter the general pattern of rapid RNA decline early in adult life, followed by steady levels at older ages. Pre- and post-DNase estimates are highly correlated, both for pooled data (r =.98; p <.001) and for lines treated separately (r =.97,.99, and.99; all p <.001).

Figure 1 suggests that the three lines have the same pattern of age-specific decline in RNA levels, in spite of dramatic differences between the lines in adult life spans. Lines also appear very similar when age-specific RNA concentrations are plotted against age expressed as a fraction of maximum life span (Figure 2). To rigorously test the hypothesis that lines have similar patterns of age-specific total RNA concentration, we executed analysis of variance (ANOVA) on the UV data, with main effects due to genotype, age, and genotype x age interaction. The error term is estimated from replicated measurements for each age and genotype, which are available only for the UV data. RNA concentrations were log-transformed for normality. Not surprisingly, the ANOVA showed that age is a highly significant factor, accounting for 64% of variance (p <.001). Line effects are not significant (p >.20) and, most importantly, line x age effects are also nonsignificant (p >.20). The lack of a significant second-order interaction term means that in each line the RNA concentration changes as a function of age in the same way. Qualitatively similar results are obtained for nontransformed data.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. RNA concentrations (µg/ml per 30 adult male Drosophila melanogaster) assayed by three methods in three inbred lines (1S9, RI7, and RI23) at 10 ages from 1–63 days posteclosion. Here adult age is expressed as a fraction of maximum life span. A, ultraviolet spectrophotometer; B, RiboGreen assay with no DNase treatment; C, RiboGreen assay with DNase; D, Agilent 2100 bioanalyzer. All lines show similar patterns of age-specific RNA decline

 
For the Agilent method, we observed six distinct molecular size peaks for the Nano ladder in all assays, as expected, reflecting the quality of the assay (Figure 3). Also, the electropherogram of all samples and all ages showed two peaks, representing the 18S and 28S ribosomal RNAs, as expected. The ribosomal RNA bands of every sample were also found to be intact by agarose gel electrophoresis (data not shown). Figure 3 shows selected electropherograms of the three lines. The absence of extra or unexpected peaks in the Agilent electropherograms suggests that the samples are free of contaminants or significantly degraded RNAs.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Representative Agilent 2100 bioanalyzer electropherograms. A, RNA 6000 ladder; B, Line 1S9, age 1 day; C, Line RI23, age 1 day; D, Line RI7, age 1 day. The RNA 6000 ladder in all assays shows six distinct peaks, reflecting the quality of the assays, and RNA samples show the two expected ribosomal peaks (18S and 28S rRNA) reflecting the integrity of the RNA samples. Only Day 1 electropherograms were shown. Other samples of all lines and all ages show similar electropherograms

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
We find that RNA concentrations estimated by three different methods in all lines are highly correlated, and that all methods indicate a dramatic decline in total RNA in the earliest samples, at ages 3 and 5 of adult life (Table 1, Figures 1 and 2). This observation agrees with previous studies on unselected Drosophila (4,10–13).

Shikama and Brack (11) proposed that the relationship between RNA levels and senescence in Drosophila could be tested by studying RNA decline in long-lived and control lines. If there were a functional relationship between RNA levels and senescence, then the dramatic decline typically observed early in adult life should be delayed in long-lived flies, compared with controls. We find no differences between genetically homogeneous lines in age-specific concentrations of RNA, suggesting that the dramatic early decline in total RNA is unrelated to patterns of senescence in the flies. The temporal pattern of change supports this view, since RNA decline ceases before mortality rates become significant (generally after 30 days). Our data clearly establish that two-fold differences in adult life spans observed in these lines are not explained by different age-specific patterns of total RNA decline.

The timing of RNA decline observed here and in some previous studies is interesting in several respects. The observed trajectory of age-specific RNA content coincides almost exactly with the trajectory of age-specific resting metabolic rates estimated by CO2 production in single flies (22); both RNA levels and metabolic rates are high shortly after emergence, and are approximately less than half their initial levels by the age of 2 weeks posteclosion. These observations suggest that a 14-day-old fly is functionally senescent. The high levels of gene transcription and translation required for metamorphosis are not present in older postmitotic adults (11), while the metabolic energy expenditures associated with mating and reproduction are typically experienced at ages less than 2 weeks in laboratory culture. The timing is striking, because even unselected inbred populations of Drosophila live much longer than 2 weeks (19), and in other kinds of populations it is not unusual to see 100-day-old flies (personal observation). Both the present study and the metabolic rate study (22) examined control and long-lived lines differing by a factor of 2 in average life spans, and in both cases longevous lines showed the same age-specific decline as controls. It appears that natural selection has "designed" laboratory-adapted flies to be genetically and metabolically active up to the age of 2 weeks, and that experimental manipulations that produce long-lived lines have not altered that fundamental program.

It was not feasible in this study to determine how aging specifically affects the pattern of synthesis of a particular RNA species (rRNA, tRNA, and mRNA). Using gel electrophoresis, we observed a decline in all RNA species at all ages in the three lines (data not shown). At the latest ages, primarily ribosomal bands were visible on agarose gel, because rRNA makes up the bulk of total RNA, while mRNA and tRNA components were barely detectable. Studies on the effect of aging on specific mRNA or protein levels will be important to better understand changes in gene expression that accompany the aging process. It has been suggested that many of these changes are due in part to mRNA decay processes (24). In mammalian cells it has been suggested that nutrient levels, hormones, cytokines, and even viral infections can alter mRNA turnover rates (25). It will be interesting to investigate the effect of some of these factors on Drosophila mRNA decay rates. It is also important to compare decay rates of flies with altered life span.

Conclusion
While our data demonstrate age-related changes in total RNA in both long-lived and control lines, the data do not support a causal role for RNA levels in explaining the differences in longevity found between the lines.


   Acknowledgments
 
We thank Dr. A. Khazaeli for collecting and maintaining flies. Research is supported by grants AG 09711 and AG 11722 from the National Institute on Aging at the National Institutes of Health.


   Footnotes
 
Decision Editor: Steven N. Austad, PhD

Received March 10, 2004

Accepted June 10, 2004


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Bal'azs A, Haranghy L. Alteration of nucleic acid content of Drosophila melanogaster imagoes during ripening and ageing. Acta Biol. 1965;15:343-350.[Medline]
  2. Lang CA, Lau HY, Jefferson DJ. Protein and nucleic acid changes during growth and aging in the mosquito. Biochem J. 1965;95:372-377.[Medline]
  3. Yale TH, Ballard RC. The determination of ribose nucleic acid in the heart and associated tissues of house flies of various ages. Comp Biochem Physiol. 1966;19:29-34.[Medline]
  4. Samis HJ, Jr, Erk FC, Baird MB. Senescence in Drosophila. 1. Sex differences in nucleic acid, protein and glycogen levels as a function of age. Exp Gerontol. 1971;6:9-18.[Medline]
  5. Ring RA. Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata. J Insect Physiol. 1973;19:481-494.[Medline]
  6. Massie HR, Baird MB, McMahon MM. Loss of mitochondrial DNA with aging in Drosophila melanogaster. Gerontologia. 1975;21:231-238.[Medline]
  7. Sharma SP, Jit I, Rai N. Age-related changes in nucleic acids and protein in Callosobruchus maculatus Fabr. (Coleoptera). Gerontology. 1984;30:26-29.[Medline]
  8. Kara TC, Patnaik BK. Some biochemical changes in the ageing heart of the male garden lizard. Arch Gerontol Geriatr. 1982;1:219-221.[Medline]
  9. Wadhwa R, Sharma SP. Changes in nucleic acid content in aging Drosophila bipectinata. Exp Gerontol. 1984;19:199-203.[Medline]
  10. Brack C, Ackermann R, Shikama N, Thuring E, Labuhn M. Drosophila as a model system for molecular gerontology. In: Rattan S, Toussaint O, eds. Molecular Gerontology: Research Status and Strategies. New York: Plenum; 1996:151–176.
  11. Shikama N, Brack C. Changes in the expression of genes involved in protein synthesis during Drosophila aging. Gerontol. 1996;42:123-136.
  12. Labuhn M., Brack C. Age-related changes in the mRNA expression of actin isoforms in Drosophila melanogaster. Gerontol. 1997;43:261-267.
  13. Brack C, Bechter-Thuring E, Labuhn M. N-acetylcysteine slows down ageing and increases the lifespan of Drosophila melanogaster. Cell Mol Life Sci. 1997;53:960-966.[Medline]
  14. Wattiaux JM, Tsien HC. Age effects on the variation of RNA synthesis in the nurse cells in Drosophila. Exp Gerontol. 1971;6:235-247.[Medline]
  15. Schwarze SR, Weindruch R, Aiken JM. Decreased mitochondrial RNA levels without accumulation of mitochondrial DNA deletions in aging Drosophila melanogaster. Mut Res. 1998;382:99-107.[Medline]
  16. Schwarze SR, Weindruch R, Aiken JM. Oxidative stress and aging reduce COX 1 RNA and cytochrome oxidase activity in Drosophila. Free Rad Biol Med. 1998;25:740-747.[Medline]
  17. Van Remmen H, Ward WF, Sabia RV, Richardson A. Gene expression and protein degradation. In: Masoro EJ, ed. Handbook of Physiology-Aging. New York: Oxford University Press; 1995:171–234.
  18. Rattan SIS. Protein synthesis and the components of protein synthetic machinery during cellular ageing. Mut Res. 1991;256:115-125.[Medline]
  19. Curtsinger JW, Khazaeli AA. Lifespan, QTLs, age-specificity, and pleiotropy in Drosophila. Mech Aging Dev. 2002;123:81-93.
  20. Lukinbill LS, Arking R, Clark MJ. Selection for delayed senescence in Drosophila melanogaster. Evolution. 1984;38:996-1003.
  21. Lukinbill LS, Clare MJ. Selection for lifespan in Drosophila melanogaster. Heredity. 1985;55:9-18.
  22. Van Voorhies WA, Khazaeli AA, Curtsinger JW. Long-lived Drosophila melanogaster lines exhibit normal metabolic rates. J Appl Physiol. 2003;95:2605-2613.[Abstract/Free Full Text]
  23. Tahoe NMA, Dean AM, Curtsinger JW. Nucleotide variations in the lxd region of Drosophila melanogaster: characterization of a candidate modifier of lifespan. Gene. 2002;297:221-228.[Medline]
  24. Brewer G. Messenger RNA decay during aging and development. Aging Res Rev. 2002;1:607-625.
  25. Guhaniyogi J, Brewer G. Regulation of mRNA stability in mammalian cells. Gene. 2001;265:11-23.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS