Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles

Abstract

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

Keywords: TRAMP complex; cell size; circadian; diurnal; feeding-fasting rhythms; liver; mouse; rRNA degradation; rRNA polyadenylation; ribosomal protein synthesis.

Figure 1. Diurnal Changes in Liver Weight Depend on Feeding Cycles

(A) Representative immunohistochemistry (IHC) images of liver sections from night-fed (top panel) and day-fed (bottom panel) mice sacrificed at 4-hr intervals around the clock. The scale bar represents 20 μm. (B and C) Diurnal oscillations of hepatocytes areas extracted from IHC images, such as the ones depicted in (A). Each time point shows the median and SEM of four independent biological samples. X, average values. For each sample, more than 1,200 cells were analyzed. (D–F) Masses of various organs at ZT0 and ZT12, expressed as percentages of total body weight, of male C57BL/6 mice subjected to light-dark cycles and fed ad libitum (D), exclusively during the night (E), and exclusively during the day (F). Data are represented as the mean ± SD for four mice per time point (D) and nine mice per time point (E and F). ***p < 0.001; ****p < 10⁻⁶; two-sided Student’s t test; n.s. means “not statistically significant“. (G) Liver weight determined in night- or day-fed animals at 4-hr intervals around the clock. The Zeitgeber times are indicated below the panel. See also Figure S1.

Figure 2. Daily Changes in Hepatic RNA and Protein Content in Mouse Liver

(A) Content in DNA, RNA, and soluble protein determined at ZT0 and ZT12 in livers of mice fed exclusively during the night or exclusively during the day. The methods used are outlined in Figures S2A and S2B and STAR Methods. (B and C) Protein/DNA ratio (B) and RNA/DNA ratio (C) in the livers of night-fed (NF) or day-fed (DF) mice at ZT0 and ZT12. The data represent the mean ± SD for eight mice per time point. Soluble protein amounts were measured by dot-blot protein assays. See also Figure S2C. (A–C) The data represent the mean ± SD for eight mice per time point (***p < 0.001; two-sided Student’s t test). (D) RNA/DNA ratio in nighttime- or day-fed mice around the 24-hr cycle. The Zeitgeber times (ZT), with ZT0, lights on, ZT12, lights off, are indicated below the panel. The data represent the mean ± SD for four to eight mice per time point. See also Figure S2.

Figure 3. Analysis of Temporal Polysome Distribution, rRNA Transcription, and rRNA Accumulation in Livers of Night-Fed Mice

(A and B) Representative polysome profiles obtained by sucrose gradient sedimentation of cytoplasmic liver extracts from mice sacrificed at ZT04 (A) and ZT16 (B). For replicates, see Figure S3. (C) 18S and 28S rRNA levels in night- (NF) or day-fed (DF) mice at ZT0 and ZT12 (see also Figure S3A). Real-time qRT-PCR quantifications of 18S and 28S rRNA from total RNA samples (extracted according to the liver whole-cell RNA protocol) were normalized to DNA content (i.e., cell number), as determined in the experiments displayed in Figure 2A. The fold differences are normalized to ZT12. The data represent the mean ± SD for six mice per time point (*p < 0.05; two-sided Student’s t test). (D) 47/45S rRNA levels in night- (NF) or day-fed (DF) mice around the clock measured by qRT-PCR and normalized to DNA content, as measured in the data shown in Figure 2A. The data represent the mean ± SD for at least three mice per time point. (E) 47/45S and 18S rRNA levels in night- (NF) or day-fed (DF) mice around the clock normalized to DNA content and analyzed by northern blot hybridization. Mice were sacrificed at 4-hr intervals (three animals/time point), RNA/DNA ratios were measured, and total RNAs were prepared and pooled. (F) 18S rRNA/47/45S pre-rRNA ratio and 47/45S pre-rRNA/DNA ratio according to the quantification of the northern blot presented in Figure 3E and the DNA content measured in Figure 2A. (G) Density of elongating RNA Pol I and Pol II molecules, around the clock. Night-fed mice were sacrificed at 6-hr intervals (two animals/time point), and liver nuclear proteins associated with chromatin were prepared. Protein extracts corresponding to 5 μg of DNA were analyzed by immunoblotting with antibodies recognizing RNA polymerase I (RPA2 subunit) and RNA polymerase II (POLR1B). Red Ponceau staining of the membrane served as loading control. See also Figure S3.

Figure 4. Polyadenylation of 18S-E rRNA Is Diurnal in the Livers of Night-Fed, but Not Day-Fed, Mice

(A) RT-PCR protocol used for the semiquantitative analysis of polyadenylated 18S-E rRNA transcripts. (B) Comparison of polyadenylated 18S-E rRNA levels around the clock in night- and day-fed mice by semiquantitative Southern blot analysis. Hybridization of RT-PCR products using a (³²P)-labeled hybridization probe specific for 18S-E rRNA (18S_1773-1802 probe) to detect 3′ polyadenylated 18S-E rRNA in mouse liver. Mice were sacrificed at 4-hr intervals (three animals/time point), and total RNAs were prepared and pooled. (C) Comparison of polyadenylated 18S-E rRNA levels around the clock in night- and day-fed mice by qRT-PCR. Night- and day-fed mice were sacrificed at 4-hr intervals, and total RNAs were prepared. The values represent mean ± SD for three mice per time point and were normalized to cyclophilin A mRNA levels. (D and E) Comparison of the levels of polyadenylated 18S-E (D) and 28S (E) rRNA at ZT04 and ZT16 in night-, day-, and ad-lib-fed mice by semiquantitative Southern blot analysis. Southern blot hybridization of RT-PCR products with an 18S rRNA-specific probe (see B; D) and a 28S rRNA-specific probe (28S_4675-4694; E) to detect 3′ polyadenylated rRNAs in mouse liver is shown. Mice (six animals/time point) were sacrificed, and total RNAs were prepared and pooled. (F) Comparison of polyadenylated 18S-E rRNA levels around the clock in wild-type (WT) and Bmal1 knockout (KO) mice, subjected to a nighttime-restricted feeding regimen, by semiquantitative analysis. Southern blot hybridization of RT-PCR products (using the hybridization probe specified in A) to detect 30 polyadenylated 18S-rRNA in mouse liver. Four to five animals per time point were sacrificed at 4-hr intervals, and total RNAs were prepared and pooled. (G and H) Comparison of polyadenylated 18S-E rRNA levels in nuclear and cytoplasmic mouse liver RNA at ZT04 and ZT16 by qRT-PCR (G) and semiquantitative Southern blot analysis (H). Night-fed mice were sacrificed at ZT04 and ZT16, liver nuclei and cytoplasmic extracts were prepared, and RNAs were prepared from these subcellular fractions. The specificity of nuclear and cytoplasmic RNA fractions were controlled by qPCR experiments (Figures S4I and S4J). The measurements of polyadenylated rRNA levels determined by qRT-PCR (G) represent means ± SD for six mice per time point and were normalized to Cyclophilin A mRNA levels (**p < 0.01; two-sided Student’s t test). For the hybridization of RT-PCR products by Southern blot hybridization (H), the RNAs from three animals per time point were pooled. (B–G) Cyclophilin A mRNA was used as a loading control, and the quantifications of the blots are shown in Figures S4D–S4G. See also Figures S4 and S7.

Figure 5. rRNAs Are Polyadenylated in the Nucleus, and Polyadenylated rRNAs Are Recognized by the 3′ -to-5′ Exosome

(A and B) Effect of RNAi using PAPD5 siRNA (siPapd5), PARN siRNA (siPARN), XRN2 siRNA (siXrn2), and EXOSC10 siRNA (siExosc10) on polyadenylated 18S-E rRNA levels in transfected NIH 3T3 cells. Polyadenylated rRNAs were measured by qRT-PCR (A) or Southern blot hybridization of RT-PCR products with the 18S-E rRNA-specific probe (B). Knockdown efficiencies were controlled by qRT-PCR (Figures S5A–S5D). (A) The data represent mean values ± SD of 18S-poly(A) rRNA levels relative to Cyclophilin A mRNA levels measured in three independent transfection experiments with non-targeting siRNAs (siCtrl) or Papd5/PARN/Xrn2/Exosc10 siRNAs. ***p < 0.001; **p < 0.01; two-sided Student’s t test. (C and D) EXOSC10 and PAPD5 protein expression around the clock in livers of night-fed animals. Night-fed mice were sacrificed at 4-hr intervals (three animals/time points), and liver nuclear proteins were prepared and pooled. Protein extracts were analyzed by immunoblotting with antibodies recognizing EXOSC10 (C), PAPD5 (D), and U2AF65 (used as a loading control; C and D). See also Figure S5.

Figure 6. Diurnal Synthesis and Nuclear Accumulation of RPs in Livers of Night-Fed Mice

(A–C) Temporal translation rates (ribosome profiling) and accumulation (RNA sequencing [RNA-seq]) of mRNAs encoding RPS3, RPS18, and RPS29. The data were extracted from an RNA sequencing and ribosome profiling analysis around the 24-hr cycle in livers of mice entrained to a nighttime feeding regimen (Atger et al., 2015). Means of four values per time point ± SD are plotted. (D, F, and H) RP levels analyzed by immunoblotting with antibodies recognizing RPS3 (D), RPS18 (F), and RPS29 (H) in nuclear liver protein extracts prepared from mice subjected to a nighttime- or daytime-restricted feeding regimen and sacrificed at ZT04 or ZT16. Immunoblotting with antibodies recognizing U2AF65 was used as a loading control. (E, G, and I) Means ± SD for six mice per time point of representative immunoblots presented in (D, F, and H) normalized to ZT04. The data represent the mean values ± SD (*p < 0.05; **p < 0.01; two-sided Student’s t test). See also Figure S6.

Figure 7. 18S-E rRNA Polyadenylation Is Enhanced by the Depletion of Ribosomal Proteins in NIH 3T3 Cells

(A and B) Effect of Rps3 siRNA (siRps3), Rps18 siRNA (siRps18), and Rps29 siRNA (siRps29) on polyadenylated 18S-E rRNA levels in transfected NIH 3T3 cells, as measured by Southern blot hybridization of RT-PCR products with 18S_1773-1802 probe (A) or by qRT-PCR (B). The knockdown efficiencies were controlled by qRT-PCR experiments (Figures S6A–S6C). (B) Data represent the mean values ± SD of polyadenylated 18S-E rRNA levels relative to Cyclophilin A mRNA levels measured in three independent transfection experiments with non-targeting siRNAs (siCtrl) or Rps3/Rps18/Rps29 siRNAs. *p < 0.05; **p < 0.01; ***p < 0.001; two-sided Student’s t test. (C) Cartoon displaying diurnal fluctuations in liver volume and hepatocyte cell size. The items in the scheme are not drawn to scale. (D) Model showing the putative roles of ribosomal protein synthesis and rRNA polyadenylation in the diurnal production of ribosomes. The daily cycle of ribosome accumulation is regulated posttranscriptionally. Whereas the synthesis of 47/45S pre-rRNA and RP mRNAs (not shown in scheme) is constant throughout the day, RP mRNAs are translated more efficiently during the activity/dark phase, during which mice feed. This leads to an imbalance between rRNA and RP production, particularly during the resting/light phase. Excess rRNAs not assembled into complete pre-ribosomal particles are polyadenylated by the TRAMP complex and degraded by the 3′ –5′ nuclear exosome. Complete pre-ribosomal subunits are exported into the cytoplasm, where they mature to functional subunits capable of mRNA translation. RNA polymerase I complexes are symbolized by light brown hexagons, and RPs associated with small and large subunits are represented as green and light brown ovals, respectively.