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Figure 4 Polyadenylation of 18S-E rRNA Is Diurnal in the Livers of Night-Fed, but Not Day-Fed, Mice Show full caption (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 3′ 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. ∗∗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. (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 S4 I 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 S4 D–S4G. See also Figures S4 and S7

Figure S4 Semiquantitative Analysis of Polyadenylated 18S-E rRNA in Liver, Related to Figure 4 Show full caption (A) Polyadenylated 18S-E rRNA levels around the clock of ad libitum fed mice by semiquantitative analysis. Hybridization of RT-PCR products (18S_1773-1802 probe) to detect 3′ polyadenylated 18S-E rRNA in mouse liver. The mice were sacrificed at 4 hr intervals (4 animals/time point) and total RNAs were prepared and pooled. Cyclophilin A mRNA was used as a loading control. (B-C) Comparison of polyadenylated 18S-E (B) and 28S (C) rRNA levels around the clock in night- and day-fed mice by quantitative RT-PCR. Night- and day-fed mice were sacrificed at 4 hr intervals, total RNAs were prepared, and polyadenylated RNAs were selected on oligodT-beads and reverse-transcribed with random hexamers. The measurements of these polyadenylated rRNA levels represent the means ± SD for 3 mice per time point normalized to Cyclophilin A mRNA levels. (D-G) Quantification of the representative Southern blots hybridizations presented in Figure 4 B (D), 4 D-E (E-F) and 4 F (G). (H) Comparison of the polyadenylated 28S rRNA levels in the nucleus and in the cytoplasm of mouse liver cells at ZT04 and ZT16, by quantitative RT-PCR. Night-fed mice were sacrificed at ZT04 and ZT16, liver nuclear and cytoplasmic extracts were prepared in parallel, and RNAs from the two compartments were prepared. The measurements of these polyadenylated rRNA levels represent means ± SD for 6 mice per time point and were normalized to Cyclophilin A mRNA levels. (∗p < 0.05 by two-sided Student’s t test). (I-J) (I) Lgals1 mRNA and pre-mRNA levels and (J) MALAT-1 RNA levels in nuclei and cytoplasm of mouse liver cells measured by quantitative RT-PCR. Night-fed mice were sacrificed at ZT04 and ZT16, liver nuclear and cytoplasmic extracts were prepared in parallel and RNAs from these two compartments were prepared. The measurements of these polyadenylated rRNA levels represent means ± SD for 12 mice and were normalized to Cyclophilin A mRNA levels. (∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by two-sided Student’s t test).

Because the oscillation in rRNA accumulation could not be attributed to diurnal rDNA transcription, rRNAs must have been diurnally degraded. Whereas rRNA transcription and processing have been extensively studied in mammals, the mechanisms involved in rRNA degradation are still poorly understood. In yeast, improperly processed pre-rRNAs are targeted through the addition of short adenylate tails by the TRAMP complex and degraded by the 3′–5′ nuclear exosome (). Polyadenylated rRNA transcripts have also been observed in mouse and human cells, possibly suggesting a similar decay pathway in mammalian cells (). If polyadenylated rRNAs were indeed targeted for degradation, the levels of polyadenylated rRNA in liver should follow a diurnal rhythm with a phase roughly opposite to that of cellular ribosome accumulation. In order to determine the dynamics of polyadenylated rRNA, we synthesized cDNA from total liver RNA collected at 4-hr intervals around the clock by using an oligo(dT)-adaptor primer and amplified the cDNAs of adenylated 18S and 28S 3′ regions with rRNA-specific forward primers and an adaptor-specific reverse primer. In these experiments, we used a low number of PCR cycles in order to keep the amplification rates within the exponential range ( Figure 4 A). The resulting PCR products were verified by sequencing. Importantly, the single PCR product obtained for polyadenylated 18S rRNA corresponded to the 18S-E precursor RNA, which is confined to the nucleus. This is in keeping with results described below, showing that rRNA polyadenylation occurs in the nuclear compartment. The amounts of polyadenylated rRNAs were quantified by Southern blot hybridization ( Figures 4 B and S4 D) and real-time qPCR ( Figure 4 C). Whereas nearly constant levels of 3′ polyadenylated 18S-E pre-rRNA were observed around the day in livers of day-fed mice, the accumulation of these transcripts followed a robust diurnal rhythm in night-fed mice, with maximal and minimal levels at around ZT04 and ZT12/ZT16, respectively ( Figures 4 B and S4 D). We also observed a similar temporal polyadenylation pattern for polyadenylated 18S-E rRNA in ad-lib-fed mice ( Figure S4 A). The accumulation of polyadenylated 28S rRNA was diurnal as well, albeit with a somewhat lower peak-to-trough ratio ( Figures 4 E and S4 F). For both polyadenylated 18S-E and 28S rRNAs, the rhythmic expression profiles were confirmed by another technique, involving the isolation poly(A)RNA by adsorption to oligo(dT)-biotin beads, reverse transcription using random primers, and quantification of the resulting cDNAs by real-time qPCR employing rRNA-specific primers ( Figures S4 B and S4C). The comparison of polyadenylated rRNA accumulation at ZT04 and ZT16 in mice subjected to the three feeding regimens demonstrated that daytime feeding abolished the daily oscillations observed in mice fed during the night or ad libitum ( Figures 4 D, 4E, S4 E, and S4F). Thus, food absorption exclusively or preferentially during the night was required not only for increasing liver mass and protein and ribosome accumulation during the activity/feeding phase but also for the antiphasic increase in the abundance of polyadenylated rRNA species that occurred during the resting/fasting phase.