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Partial Inhibition of RNA Polymerase I Promotes Animal Health and Longevity

longevity rna polymerase i aging ribosomal rna/dna drosophila old-age health lifespan

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#1 Engadin

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Posted 15 February 2020 - 01:17 PM


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F U L L   T E X T   S O U R C E :    Cell

 

 

 

 

 

Highlights

 
  •  Partial inhibition of RNA polymerase I (Pol I) can extend lifespan in the fruit fly
 
  •  Reducing Pol I activity after development and only in the gut is sufficient
 
  •  Pol I activity affects aging from both post-mitotic and mitotically active cells
 
  •  Pol I activity affects the age-related decline in performance of multiple organs
 
 
Summary
 
Health and survival in old age can be improved by changes in gene expression. RNA polymerase (Pol) I is the essential, conserved enzyme whose task is to generate the pre-ribosomal RNA (rRNA). We find that reducing the levels of Pol I activity is sufficient to extend lifespan in the fruit fly. This effect can be recapitulated by partial, adult-restricted inhibition, with both enterocytes and stem cells of the adult midgut emerging as important cell types. In stem cells, Pol I appears to act in the same longevity pathway as Pol III, implicating rRNA synthesis in these cells as the key lifespan determinant. Importantly, reduction in Pol I activity delays broad, age-related impairment and pathology, improving the function of diverse organ systems. Hence, our study shows that Pol I activity in the adult drives systemic, age-related decline in animal health and anticipates mortality.
 
 
fx1_lrg.jpg
 
 
 
 
Introduction
 
Most animals age; their vitality, health, and survival decline as they get older (Martínez, 1998). In humans, age is the main risk factor for the predominant killer and debilitating diseases, including cancer, cardiovascular disease, and neurodegeneration (Christensen et al., 2009, Fontana et al., 2010, López-Otín et al., 2013, Niccoli and Partridge, 2012). Because the proportion of older people in many populations is increasing at an alarming rate (Christensen et al., 2009), identifying mechanisms that can be harnessed to improve human health and well-being in old age is an urgent priority for biomedical research. Over the last three decades, research in basic biogerontology has shown that aging is moldable by identifying fundamental cellular and organismal processes that can be manipulated to extend a healthy lifespan (Alic and Partridge, 2011, López-Otín et al., 2013, Partridge et al., 2018). These processes can often be altered in the adult to achieve longevity by reprogramming gene expression via transcriptional regulation.
 
The task of transcription in the eukaryotic nucleus is divided between three nuclear RNA polymerases (Pols) (Roeder and Rutter, 1969). Pol I, Pol II, and Pol III have distinct subunit composition, have distinct biochemical properties, and transcribe distinct classes of genes (Roeder and Rutter, 1969, Werner and Grohmann, 2011, Vannini and Cramer, 2012). Pol II-generated transcripts include all protein-coding messenger RNAs (mRNAs) and perform a vast number of cellular functions. The historical focus on Pol II is also observed in aging research, in which the most effort has been invested in understanding how a number of Pol II transcription factors guide pro-longevity transcriptional programs (e.g., Murphy et al., 2003, Hsu et al., 2003, Tepper et al., 2013, Alic et al., 2011, Alic et al., 2014, Dobson et al., 2019). The other two Pols have received much less attention, despite their fundamental cellular roles. We have recently described an evolutionarily conserved role for Pol III in aging (Filer et al., 2017). However, the role of Pol I remains unexplored.
 
Pol I is the fundamental structurally and functionally conserved eukaryotic enzyme that transcribes a single gene, ribosomal DNA (rDNA) (Vannini and Cramer, 2012). It generates the pre-rRNA that is processed into the mature 18S, 5.8S, and 28S rRNAs, the key structural and catalytic components of the ribosome (Grummt, 2003, Kressler et al., 2017). Even though Pol I has only one task to perform, its activity accounts for a major portion of cellular transcription due to the high cellular demand for rRNA (Grummt, 2003). rDNA is present in hundreds of copies per genome that are organized in large arrays of tandem repeats in one or several genomic locations (Grummt, 2003). Pol I is known to be essential for cellular growth and proliferation and for organismal growth, and it is often deregulated in cancers (Drygin et al., 2010, Grewal et al., 2007, Ghosh et al., 2014, Sriskanthadevan-Pirahas et al., 2018). The focus on this growth-promoting role of Pol I has often precluded investigating its potential function(s) in more complex cellular and animal traits. However, recent work has generated unsuspected insights. For example, Pol I activity in one tissue can promote organismal growth via secreted factors (Ghosh et al., 2014); it can equally affect not only cell proliferation but also cell fate decisions in a stem cell lineage (Zhang et al., 2014). Here, we present evidence that Pol I activity itself is a central cause of aging, affecting survival as well as multiple indices of health in the animal model Drosophila melanogaster.
 
 
Results
 
Pol I Activity in the Adult Limits Animal Lifespan
 
Pol I is composed of 14 subunits, several of which are specific to the enzyme (Vannini and Cramer, 2012, Fernández-Tornero et al., 2013, Engel et al., 2013). To examine aging in a Pol I loss-of-function mutant, we backcrossed a transposon insertion in the 5′ region of the Rpl1 gene (P-element, SH0507) encoding the largest specific subunit of Pol I (ortholog of the A190 subunit of budding yeast [Figure S1A] and the human POLR1A) into a healthy, outbred, wild-type fly background. The RpI1SH0507 allele (RpI1SH henceforth) is recessive, pre-adult lethal. The viable RpI1SH/+ adult heterozygote females had a 50% reduction in RpI1 mRNA (p < 10−4; Figure 1A), while not showing a significant effect on the expression of the neighboring gene Blos1 (p = 0.13; Figure S1B), confirming a substantial and specific effect of the insertion on the expression of RpI1. Pol I activity is essential for animal growth, which in Drosophila occurs during the larval stages (Grewal et al., 2007). RpI1SH/+ females did not weigh substantially less than controls (Figure S1C), indicating that the heterozygous mutation does not impose a major growth impairment.
 
 
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Figure 1. Reduction in Pol I Activity Extends Fruit Fly Lifespan
 
(A)   Relative RpI1 transcript levels (n = 8 biologically independent samples, p < 10−4, t test).
 
(B)   Ratio of RNA to DNA for the sequences present in pre-rRNA-rDNA (n = 8 biologically independent samples, effect of genotype p = 9 × 10−4, no significant effect of the target sequence or interaction, linear model [LM]).
 
©   Relative levels of protein synthesis in whole flies determined by puromycin incorporation and western blotting, showing a representative blot (left) and quantification (right; n = 5 individual flies, p = 0.01, t test).
 
(D)   Representative images of nucleoli (Fibrillarin staining) in posterior midguts (scale bar, 10 μm).
 
(E)   Quantification of nucleolar size in large or small nuclei (area as proportion of nuclear area; n = 1–5 nucleoli per animal per nuclear size, 4–5 animals where values from the same animal are indicated as vertically aligned points in the boxplot, effect of genotype p = 0.01, nuclear size p < 1 × 10−4, interaction p = 2 × 10−4, mixed-effects LM with “animal” as random effect).
 
(F)   Lifespans of RpI1SH/+ (n = 137 dead/15 censored flies), Tif-1AKG/+ (n = 118 dead/25 censored flies) and wild-type females (n = 118 dead/22 censored flies, p = 3 × 10−4 and 1 × 10−4, respectively, log-rank test).
 
(G)   Lifespans of females after ubiquitous induction of dA43RNAi from day 2 of adulthood by RU486 feeding and controls (−RU486 n = 154 dead/4 censored flies, +RU486 n = 155 dead/1 censored flies, p = 4 × 10−12, log-rank test).
 
(A)–(E)   were assessed in RpI1SH/+ and wild-type females. Boxplots show means and quintiles, with individual replicate points overlaid.
 
See also Figures S1 and S2.
 
 
 
 
To examine whether the RpI1SH/+ adults exhibit phenotypes consistent with partial Pol I inactivation, we examined Pol I activity, rates of protein synthesis, and nucleolar size in the mutants. To characterize Pol I activity, we determined the relative RNA to DNA abundance of sequences present in the pre-rRNA—within the 5′ externally transcribed spacer (5′ETS), internally transcribed spacer (ITS), and the mature 18S and 28S rRNA. A 30% overall reduction in this rRNA/rDNA ratio was observed in RpI1SH/+ adult females (p = 9 × 10−4 Figure 1B), confirming a partial loss of Pol I activity in vivo. Using puromycin incorporation assays,we found that this reduction in rRNA synthesis was accompanied by a reduction in the rates of protein synthesis in individual,whole RpI1SH/+ females (p = 0.01; Figures 1C and S1D).
 
Pre-rRNA transcription and ribosome biogenesis take place in the nucleolus, a subnuclear compartment whose formation is seeded by the rDNA repeats (Grummt, 2003). Nucleolar size is indicative of the levels of ribosome biogenesis (Tiku and Antebi, 2018, Tiku et al., 2017, Uppaluri et al., 2016). To investigate the cellular consequences of the reduction in Pol I activity in RpI1SH/+ females, we examined the nucleolar size of the Drosophila midgut (equivalent to the mammalian small intestine). This organ harbors cells with a large nucleus, the absorptive, post-mitotic enterocytes (ECs), and two types of cells with smaller nuclei, the enteroendocrine cells and the mitotically active intestinal stem cells (ISCs) (Miguel-Aliaga et al., 2018). We assessed whether the relative nucleolar size in gut cells was altered in RpI1SH/+ females by staining for the nucleolar marker Fibrillarin and analyzing the data with a mixed-effects linear model (LM) to account for different nuclear sizes and intraindividual correlation. We found that RpI1SH/+ females displayed a reduction in relative nucleolar area compared to controls (p = 0.01; Figures 1D and 1E). This reduction was greatest in ECs (genotype-by-nuclear size interaction p < 10−4; for a summary of the LM analysis, see Figure S1E). This was possibly due to the higher demand for protein synthesis in ECs, as indicated by their larger relative nucleolar size (Figures 1D and 1E). We did not observe a substantial difference in nuclear size or ploidy (measured as DAPI staining intensity) between the genotypes (Figures S1F and S1G), which is consistent with a limited effect of the heterozygote mutation on fly growth (Figure S1C). In summary, a heterozygous loss-of-function mutant in the gene encoding the largest subunit of Pol I is viable and displays molecular and cellular phenotypes that are consistent with a partial reduction in Pol I activity. We next sought to examine how this reduction in Pol I activity affects aging.
 
To characterize the role of Pol I activity in aging, we focused on females, whose aging is more malleable and better characterized. We found that RpI1SH/+ females lived longer than the wild-type controls (Figure 1F). To determine the statistical significance of this observation, we used the log-rank test, which assesses the difference in survival across the lifespan and is highly sensitive. RpI1SH/+ females were significantly longer lived than the wild-type (p = 3 × 10−4). To confirm that this longevity is due to a reduction in Pol I activity, we used additional, independent genetic reagents. We backcrossed the previously characterized Tif-1AKG06857 allele (Tif-1AKG henceforth), which abolishes almost completely the expression of the essential activator of Pol I, Tif-1A, resulting in a reduction in Pol I activity (Grewal et al., 2007). Tif-1AKG/+ females also showed a significant lifespan extension (p = 1 × 10−4; Figure 1F). Furthermore, the longevity of both RpI1SH/+ and Tif-1AKG/+ females was robustly observed in three independent experimental trials with an average 8% extension of the median lifespan (Figure S1H). Hence, partial lifelong reduction in Pol I activity extends lifespan.
 
As Pol I is crucial for fly development (Grewal et al., 2007, Ghosh et al., 2014), the longevity observed in the RpI1SH/+ or Tif-1AKG/+ females could have resulted from a developmental effect of reduced Pol I activity, such as reduced growth, that influenced the subsequent adult lifespan. To examine the consequence of reducing Pol I activity specifically in adulthood, we targeted either Tif-1A or the Drosophila gene encoding the Pol I-specific subunit A43 (dA43, CG13773; Figure S1A), with RNA interference (RNAi) in combination with inducible, ubiquitous Actin- or daughterless-GeneSwitch drivers (ActGS or daGS, respectively) (Osterwalder et al., 2001). The efficacy of the Tif-1ARNAi line has been demonstrated (Ghosh et al., 2014), and we further confirmed that the ubiquitous expression of either the dA43RNAi or Tif-1ARNAi construct during development resulted in lethality, as expected (Figure S2A). Their ubiquitous induction in adulthood, achieved by feeding ActGS>dA43RNAi or daGS>Tif-1ARNAi females with the RU486 inducer from day 2 post-eclosion, was enough to extend lifespan (p = 4 × 10−12 for Figure 1G; see also Figures S2B and S2C). These data indicate that the developmental roles of Pol I, such as promoting growth, are separable from the role of Pol I in longevity, as is the case for the growth-promoting insulin/insulin-like growth factor signaling pathway (Clancy et al., 2001). In addition, RU486 feeding did not have a significant effect on the lifespans of the driver- or transgene-alone controls (Figures S2D–S2G). Note that we tested a range of RU486 doses and found that higher doses of the inducer produced a diminished or negative effect (Figures S2B and S2C), indicating that an extensive reduction in Pol I activity may be detrimental. Taken together, these experiments reveal that Pol I activity in the adult limits animal lifespan.
 
 
Pol I Activity in Multiple, Distinct Adult Cell Types Affects Organismal Aging
 
Pol I could limit lifespan from discrete subsets of adult cells. To map where its activity is relevant to longevity, we induced dA43RNAi or Tif-1ARNAi constructs using a panel of tissue- or cell-specific GeneSwitch drivers. Driving the dA43RNAi or Tif-1ARNAi constructs in the fat body and midgut (the former being equivalent to mammalian adipose tissue and liver) with the S1106 driver or in neurons with elavGS showed modest effects, significantly extending lifespan in 2 of 3 and 1 of 2 experimental trials, respectively (p = 6 × 10−3, 2 × 10−4, and >0.05; Figures 2A, S3A, and S3B; p = 0.01 and >0.05, Figures 2B and S3C), indicating some, albeit moderate, relevance of these tissues. The induction of dA43RNAi in the fly muscle with MHCGS was detrimental (Figure S3D). This adverse effect may reflect a high requirement for protein synthesis to maintain muscle function. In contrast, knocking down Pol I transcriptional machinery in the midgut with the midgut-restricted TIGS driver (Poirier et al., 2008) consistently extended lifespan (p < 0.01; Figures 2C and S3E). We confirmed the expected reduction in the pre-rRNA:rDNA ratio in the midgut of TIGS>dA43RNAi females (p = 1 × 10−4; Figure 2D). Overall, the survey of Drosophila tissues indicated that the main longevity effect of Pol I inhibition stems from the midgut, with possible minor contributions from fat body cells and neurons.
 
 
gr2_lrg.jpg
 
 
 
Figure 2. Tissue- and Cell-Type-Restricted Inhibition of Pol I Promotes Longevity
 
(A)   Lifespans of females with dA43RNAi induced in adult fat body and gut by RU486 feeding and controls (−RU486 n = 118 dead/17 censored flies, +RU486 n = 127 dead/12 censored flies, p = 6 × 10−3, log-rank test).
 
(B)   Lifespan of females with adult-onset induction of dA43RNAi in neurons achieved by RU486 feeding (−RU486 n = 138 dead/1 censored flies, +RU486 n = 140 dead/1 censored flies, p = 0.01, log-rank test).
 
©   Lifespans of females with dA43RNAi induced in the adult gut by RU486 feeding and controls (−RU486 n = 149 dead/4 censored flies, +RU486 n = 148 dead/2 censored flies, p = 7 × 10−3, log-rank test).
 
(D)   Ratio of RNA to DNA for the sequences present in pre-rRNA-rDNA in fly guts after adult-onset induction of dA43RNAi (n = 4 biologically independent samples, effect of RU486 p < 10−4, no significant effect of the target sequence or interaction, LM). Boxplots show means and quintiles, with individual biological replicate values overlaid as points.
 
(E)   Lifespans of females with dA43RNAi induced in adult ECs by RU486 feeding and controls (−RU486 n = 135 dead/8 censored flies, +RU486 n = 138 dead/9 censored flies, p = 1 × 10−13, log-rank test).
 
(F)   Lifespans of females with dA43RNAi induced in adult ISCs by RU486 feeding and controls (−RU486 n = 134 dead/2 censored flies, +RU486 n = 139 dead/5 censored flies, p = 6 × 10−6, log-rank test).
 
Fly genotypes are indicated in each panel. See also Figures S2 and S3.
 
 
 
The Drosophila midgut contains several cell types. Driving dA43RNAi in the post-mitotic ECs or mitotically active ISCs was sufficient to extend lifespan (GS5966 and GS5961 drivers, respectively, p < 10−5; Figures 2E and 2F). In all of the cases, RU486 feeding had no effect on the lifespans of driver-alone controls (Figures S3F–S3H). Overall, our data revealed that Pol I activity drives aging from distinct adult cell types. Pol I acts non-redundantly from both post-mitotic cells, such as ECs, and cells with a proliferation potential, the ISCs. We next focused on the ISCs due to their importance in gut homeostasis (Miguel-Aliaga et al., 2018) and the known role of Pol III in these cells (Filer et al., 2017).
 
 
rRNA Biogenesis in the ISCs Limits Lifespan
 
Ribosome biogenesis is emerging as an important process in the regulation of stem cell behaviors (Zhang et al., 2014, Sanchez et al., 2016). Reduction in Pol I activity in the ISCs may extend lifespan by limiting the provision of rRNAs required for ribosome biogenesis. While Pol I is the only polymerase that transcribes the rDNA locus, it is not the only polymerase that synthesizes rRNA species; 5S rRNA is generated by Pol III (Figure 3A). We have recently shown that Pol III activity in the gut also limits the lifespan in Drosophila (Filer et al., 2017). The fly midgut displays sexually dimorphic physiology and aging (Regan et al., 2016, Hudry et al., 2016, Hudry et al., 2019), and similar to the effects of Pol III, inhibition of Pol I in the midgut did not extend lifespan in males (Figure S4A). A further similarity between the effects of Pol I and Pol III is that each polymerase limits lifespan from the ISCs (see Figure 2F and Filer et al., 2017). This highlights the ISCs as the midgut cells in which both Pol I and Pol III are relevant to lifespan and highlights rRNA synthesis in the ISCs as a mechanism of longevity downstream of Pol I and Pol III, since the one task shared by both Pols is to provide the full, requisite complement of rRNA species (Kressler et al., 2017).

 

 

gr3_lrg.jpg

Figure 3. Pol I and Pol III Act in the Same Longevity Pathway in the ISCs

 

(A)   Contribution of each Pol to ribosome biogenesis. rRNAs are indicated in red. RP, ribosomal protein.

 
(B)   Lifespans of females with dA43RNAi alone (−RU486 n = 138 dead/6 censored flies, +RU486 n = 137 dead/5 censored flies, p = 0.01, log-rank test) or together with dC160RNAi (−RU486 n = 99 dead/32 censored flies, +RU486 n = 127 dead/10 censored flies, p = 2 × 10−5, log-rank test) induced in adult ISCs by RU486 feeding and controls. The lifespans of the two −RU486 or the two +RU486 conditions are not significantly different.
 
See Figure S4 for GS5961>dC160RNAi lifespans that were carried out in parallel and the CPH analysis.
 
 
 
 
To further examine the connection between rRNA synthesis and longevity, we directly assessed the genetic interactions between Pol I and Pol III activity in the ISCs for lifespan. If the two Pols do not act through a common longevity mechanism, we would expect the effects of reducing their activity on lifespan to be additive. We found that inducing the expression of a validated RNAi line against the largest Pol III subunit, dC160 (Filer et al., 2017), did not further extend lifespan when co-expressed with Tif-1ARNAi in the ISCs (Figure 3B), while it did when expressed alone in a parallel experiment (Figure S4B). Note that the presence of UAS-Tif-1ARNAi did not appear to hamper the ability of the GS5961 driver to induce a second transgene (Figure S4C). For a robust analysis of survival data, we used a Cox proportional hazards (CPH) model, which can assess the statistical significance of individual effects (genotype and presence of RU486) and of their interaction to determine whether the lifespan response to RU486 is altered by the presence of both RNAi lines. CPH analysis confirmed that the lifespan extension obtained by reducing Pol I or Pol III activity individually in the ISCs was not significantly different from that observed when both were targeted simultaneously (Figure S4D). This result is consistent with Pol I and Pol III acting in the same longevity pathway in the ISCs. This conclusion is additionally supported by the known roles of the two polymerases in the same process, namely ribosome biogenesis, and the similar profile of organs whence their activity affects lifespan. While it may be tempting to view one Pol as upstream of the other, such a cascade is unlikely since the three nuclear polymerases appear tightly coordinated for ribosome biogenesis (Martin et al., 2006, Filer et al., 2017, Grummt, 2003).
 
 
Pol I Activity Defines a Central Aging Process with Broad Effects on Health
 
Having established that a moderate reduction in Pol I activity was sufficient to extend lifespan, we next sought to examine how Pol I activity affects health in old age. Since the midgut appeared critical for longevity, we initially focused on the health and integrity of this organ.
 
 
 
 
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Also tagged with one or more of these keywords: longevity, rna polymerase i, aging, ribosomal rna/dna, drosophila, old-age health, lifespan

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