Lifelong persistence of nuclear RNAs in the mouse brain

Abstract

Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type-specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.

Fig. 1. Long-term retention of RNA in the mouse brain.

(A) Schematic diagram of experimental plan. RNA synthesis at P3-P5 was labeled using 5-Ethynyl-uridine (EU). (B) EU signals in the dentate gyrus (DG) of a 1-week-old animal. (C) EU signals in the cerebellum (CB) of a 1-week-old animal. (D) EU signals in the DG of a 1-year-old animal. (E) EU signals in the CB of a 1-year-old animal. (F-H) High magnification images with Sox2/GFAP immunostaining (arrowhead, RGL-ANSC; open arrowhead, astrocytes) or NeuN/GFAP staining (arrowheads, NeuN⁺ neurons). (J) Quantification of the density of EU⁺ cells. 1W (1-week-old) DG 3933 ± 911.9 cells/mm²; 1W CB 6060 ± 774.3 cells/mm²; 1W S1 291.8 ± 52.12 cells/mm². 1Y (1-year-old) DG 4156 ± 542 cells/mm²; 1Y CB 4816 ± 537 cells/mm²; 1Y S1 359 ± 199 cells/mm²; NS not significant; ANOVA with post-hoc Tukey’s test. Data are presented as mean +/- standard deviation (SD). (K) Quantification of the percentage of EU⁺ neurons (NeuN⁺ cells). (L) EU signal in neurons of the DG in a 2-year-old animal. (M) EU signals in a neural stem cell (RGL-ANSC) in the DG of a 2-year-old animal. Data are presented as mean +/- SD. Scale bars, 100 μm (B, D), 10 μm (F-I, L, M), 200 μm (C, E). ANOVA followed by Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.

Fig. 2. Molecular identity of long-retained RNA.

(A) Retention of EU signals in quiescent NPCs (quiNPCs) in vitro. Proliferating NPC (proNPC). Scale bar, 10 μm. (B) Quantification of EU intensity in proNPCs and quiNPCs at day 1 (D1) or day 8 (D8) after EU labeling. Kruskal-Wallis test followed by Dunn’s multiple comparison test; ***P < 0.001, ****P < 0.0001, dots indicate individual cells. (C) MA plot showing EU-enriched (gene-derived) RNAs in quiNPCs at 8 days after EU labeling, as determined by EU-RNA-Sequencing. Significantly EU-enriched RNAs were defined as adjusted P < 0.05 and fold change > 2 compared to non-EU-labeled quiNPCs (n=3 experiments). (D) Gene class distribution of EU-enriched RNA in quiNPCs. (E) Protein-coding RNAs were underrepresented, whereas long non-coding RNAs (lncRNA) and uncharacterized transcripts (TEC) were over-represented in EU-enriched RNA. ***P < 0.001 (linear regression). (F) Top significantly enriched Reactome pathways among EU-enriched RNAs in quiNPCs (adjusted P < 0.05). (G) MA plot showing EU-enriched (gene-derived) RNAs in hippocampus tissue of 5-week-old mice that were injected with EU at postnatal days P3-P5 (n = 3 mice/group). (H) Gene class distribution of EU-enriched RNA in the hippocampus. (I-J) Distribution of EU-enriched RNAs that map to repeat RNAs in quiNPCs and hippocampus tissue.

Fig. 3. Repeat RNAs are long retained in the mouse brain.

(A and B) Localization of EU signals in nuclei in the DG and CB of a 1-year-old animal. Scale bars, 5 μm. (C and D) Enrichment of EU-labeled transcripts in the hippocampus/cortex (HC/CX) of a 1-week-old animal (n = 5) and in the hippocampus (HC) of 24-week-old to 1-year-old animals (n = 4). One sample t-test, *P < 0.05, **P < 0.01. Fold enrichments compared to PBS-treated control: 1W Gapdh, 4.95 ± 1.85 ; 18S rRNA 3.76 ± 1.43; 28S rRNA 2.84 ± 1.13; major satellite 7.69 ± 4.16; minor satellite 4.48 ± 2.71; βactin 4.73 ± 2.54; SINEB1 1.95 ± 0.60; LINE-1 2.47 ± 0.62; n = 5 ; 24W-1 Y, Gapdh, 1.85 ± 0.66; 18S rRNA 2.38 ± 1.68; 28S rRNA 1.21 ± 0.63; major satellite 4.57 ± 1.55; minor satellite 4.54 ± 2.37; β actin 1.60 ± 0.68; SINEB1 1.44 ± 0.70; LINE-1 1.90 ± 1.34; n = 4. Data are presented as mean +/- SD. (E) Enrichment of EU-labeled transcripts in quiNPCs 8 days after EU treatment compared with PBS-treated cells. One sample t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Fold enrichments compared to control: Gapdh, 3.53 ± 1.50; 18S rRNA 6.86 ± 2.06; 28S rRNA 7.38 ± 3.33; major satellite 49.3 ±19.1; minor satellite 52.57 ± 20.97; β actin 22.36 ± 10.2; SINEB1 40.45 ± 25.53; LINE-1 59.82 ± 47.95; n = 6. Data are presented as mean +/- SD. MajSat, major satRNA; MinSat, minor satRNA. (F) Quantification of EU signal intensity after LNA-GapmeR-mediated knock-down of major satRNAs in quiNPCs (LNA MajSat). ****P < 0.0001, t-test, dots indicate individual cells from 3 experiments. (G) Reduction of EU signals in quiNPCs after the application of LNA targeting major satRNAs. Scale bar, 20 μm. (H-I) Increased EU signals after CRISPRa-based overexpression of major satRNAs (sgRNA MajSat). ****P < 0.0001, t-test, dots indicate individual cells from 3 independent experiments. Scale bar, 20 μm.

Fig. 4. Major satRNAs are essential for maintenance of NPC function.

(A) Distribution of H3K9me3 after LNA-mediated knock-down of major satRNAs in quiNPCs. Scale bar, 5 μm. (B) Quantification of H3K9me3 intensity after knock-down of major satRNAs. **P < 0.01, t-test, dots indicate individual cells from 3 experiments. (C) Distribution of H3K9me3 after CRISPR-mediated transcriptional inhibition (CRISPRi) or overexpression (CRISPRa) of major and minor satRNAs. Scale bar, 5 μm. (D) Transcriptional inhibition of major satRNAs using CRISPRi (dCas9-KRAB with sgRNAs) impaired heterochromatin retention in quiNPCs; minor satRNAs were dispensable. ***P < 0.001, n. s. not significant, t-test, dots represent individual nuclei from 3 independent experiments, group means are indicated. (E-F) LNA-mediated knock-down of major satRNAs reduced H3K9me3 abundance at major and minor satellite repeats. Shown are data from H3K9me3-ChIP-qPCR (*P < 0.05; one-sided t-test, n = 4). Data are presented as mean +/- SD. (G) Upregulation of satRNAs increased nuclear H3K9me3 in quiNPCs. ****P < 0.0001, t-test, dots indicate individual cells from 3 independent experiments, group means are indicated. (H) Experimental timeline for data shown in (I-N). (I) Cell cycle re-entry of quiNPCs after LNA-mediated knock-down of major satRNAs. Arrows indicate pyknotic cells. Scale bar, 25 μm. (J-K) Decreased percentage of Ki67⁺ and BrdU⁺ cells one day after re-activation of quiNPCs into proliferation. *P < 0.05, **P < 0.01, paired t-test, n = 5-8. Ki67, LNA control 23.1 ± 2.73%, LNA Majsat 11.8 ± 4.16%; BrdU, LNA control 29.6 ±12.35%, LNA Majsat 19.65 ± 13.64%. (L) Increased percentage of pyknotic cells after knock-down of major satRNAs. LNA control 13.4 ± 2.55%, LNA Majsat 22.76 ± 8.11%. (M-N) Increased percentage of γH2AX⁺ cells after knock-down of major satRNAs. *P < 0.05, ratio paired t-test, n = 4. LNA control 8.57 ± 3.25%, LNA Majsat 30.9 ± 6.66%. Scale bar in M, 5 μm.