Long Live the RNAs: The Guardians of Neuronal Longevity?

SUMMARY

In a recent publication in Science, Zocher et al.¹ identify and characterize long-lived nuclear RNA in the mouse brain, suggesting their potential roles as guardians of neuronal longevity.

Highly variable and tightly regulated, RNA turnover is the norm of the RNA life cycle. Most RNAs have relatively short lifespans, from minutes to hours, depending on the specific RNA molecules, cellular conditions, and regulatory mechanisms at play². Until recently, a half-life on the order of weeks was considered rare, associated only with very “stable” RNA³. However, Zocher et al.¹ have unveiled the existence of extremely stable RNAs persisting for months, even years, in the mouse brain, which they have named long-lived RNA (LL-RNA).

Incorporation of uridine analog 5-ethynyluridine (5-EU) is widely used to label and isolate newly synthesized RNA molecules in cells or tissues. To investigate RNA stability in the adult mouse brain, Zocher et al.¹ employed EU pulse labeling via subcutaneous injection over three consecutive early postnatal days, followed by click chemistry to visualize the EU-labeled RNA transcripts at later times (Figure 1A). They also sequenced EU-enriched RNAs in the hippocampus and quiescent neural progenitor cells (NPCs), revealing both protein-coding and noncoding RNA species. Notably, long noncoding RNAs (lncRNAs), as well as repetitive elements such as small nuclear RNAs (snRNAs), short interspersed nuclear elements (SINEs), and satellite RNAs are significantly enriched.

Figure 1. The molecular characteristics and roles of cell-specific nuclear LL-RNAs in the mouse brain.

(A)Long-lived RNAs (LL-RNAs) persist over years in the dentate gyrus (DG) and cerebellum (CB) after pulse EU labeling for 3 continuous early postnatal days. Both coding and noncoding LL-RNAs are predominantly found within the nuclei of adult neural progenitor cells (ANPCs) and neurons. Major satellite RNAs (satRNAs) maintain the organization and integrity of heterochromatin marked by trimethylated histone 3 lysine 9 (H3K9me3) in the quiescent NPC (quiNPC) model and are essential for preserving NPCs. (B) One mechanism of neural-specific, RNA-dependent regulation of longevity involves alternative splicing coupled with non-sense mediated mRNA decay (AS-NMD) of Bak1 mRNA transcripts to reduce BAK1 protein expression and attenuate apoptosis competence. PTC: premature termination codon. Whether and how LL-RNAs contribute to neuronal longevity remains to be determined. This figure was created with Biorender.com.

Pathway enrichment analysis pointed to potential functions for LL-RNAs in nuclear architecture, epigenetic regulation, and chromatin organization. One example highlighted by Zocher et al. is the critical role of repetitive RNAs, particularly major satellite RNAs, in maintaining heterochromatin integrity and NPC function. Loss- and gain-of-function analyses demonstrated the essential role of major satellite RNAs in maintaining heterochromatin organization in quiescent NPCs, as evidenced by the disorganized and altered H3K9me3 signals (Figure 1A). Furthermore, knockdown of major satellite RNAs had detrimental effects on proliferation, apoptosis, and DNA integrity in NPCs. However these experiments manipulated the levels and transcription of the satellite RNAs not their intrinsic high stability per se, for which one would need stability-altering mutants, ideally in combination with transcriptional compensation to maintain overall RNA levels.

The recognition of LL-RNA based on EU enrichment makes the assumption that EU, once incorporated during the initial injection period, remains stably incorporated. While this assumption may be reasonable, the extraordinary observation warrants further validation. Could 5-EU monophosphates, derived from RNA degradation, be used to regenerate 5-EU triphosphates for nascent RNA synthesis and are certain neurons particularly efficient in this nucleoside salvage pathway? If so, recycled EU could be incorporated into late-generated RNA species instead of early-transcribed RNA. EU is only a transcriptional label and does not necessarily signal the absence of RNA decay. Furthermore, could excessive incorporation of EU, an unnatural uridine analog, render immunity from RNA decay to certain transcripts, due to sequence dependent modifications or structural alterations?

To contest these alternative hypotheses, one can examine the residual RNA at early and late time intervals after turning off RNA synthesis. The authors followed this idea by treating quiescent NPCs with a RNA polymerase inhibitor, albeit for only one day, after EU labeling. To bolster their findings, transcriptional inhibition of specific LL-RNAs using spatiotemporal-controlled CRISPRi or CRISPRko would allow direct evaluation of the residual LL-RNAs in vivo over a longer period, using techniques like RNA sequencing or RT-qPCR; this has not yet been undertaken.

Nevertheless, the observed EU signals did display a high degree of specificity in RNA identities, brain regions, cell types, and age-dependency. Nuclear lncRNA and repeat RNA were particularly enriched, raising intriguing questions about their retention and protection in neuronal nuclei. Do they deploy RNA modifications⁴, RNA-binding proteins⁵, or the associated chromatin structures to increase stability and protect them from degradation? Or are they natively resistant to RNases thanks to specific tertiary structure configuration? Or are there high-order organizations, liquid droplets, or condensates involved in shielding them from decay? Since nuclear lncRNAs as a population are generally less stable than other RNAs⁶, investigating the mechanisms underlying the specificity of LL-RNA is poised to derive novel insights about RNA stability controls.

The EU signals were found mostly in the nuclei of NeuN+ neurons and Sox2+ adult neural progenitor cells in the dentate gyrus and cerebellum, with minimal detection in other cell types or brain areas. This nuclear enrichment may not be surprising given the close association between RNA degradation and translation in the cytoplasm^7,8. However, the mechanism governing the region and cell type specificity of LL-RNAs remains a mystery. Future investigation should aim to distinguish between two possibilities: specific gain of EU signals in these cell types vs selective protection of EU-labeled RNA from degradation.

More intriguingly, there seems to be a “birth” window of LL-RNAs, characterized by observable EU signals only if pulse labeling within a specific age frame (postnatal day 3 to 15). EU labeling during early embryonic day 14–16 or postnatal day 25–27 resulted in negligible EU signals. Although LL-RNAs tend to be detected in progenitor cells, NPCs are quite abundant in the embryonic brain, so their presence alone is insufficient to explain this phenomenon. One possibility worth investigation is the correlation with the emergence of quiescent neural stem cells. Notably, this “birth” window also resembles the slightly wider critical period of brain development, during which the brain exhibits a heightened plasticity or capacity to reorganize in response to external stimuli⁹. Whether these two temporal windows are intrinsically linked is unknown. Re-examining the “birth” window of LL-RNAs upon perturbation of the critical period may give some clues.

The most important question raised from the findings by Zocher et al. is the relationship between LL-RNAs and neuronal longevity (Figure 1B). The extreme stability of LL-RNAs, once integrated into heterochromatin, may merely mirror neuronal longevity; their functions may not require resistance to RNA degradation. Alternatively, some LL-RNAs, acting akin to the Titan Atlas, may be permanently bound to a crucial molecular task where their degradation or even momentary removal risks catastrophic consequences including neuronal death. There are RNA-based mechanisms ensuring minimal chance of cell death in neurons (e.g., via neural-specific splicing of a microexon in pro-apoptotic gene Bak1¹⁰). Even heterozygous removal of the Bak1 microexon is sufficient to re-expose neurons to a heightened apoptosis risk and leads to perinatal lethality in mice. Therefore, neurons do employ neural-specific RNA-dependent regulatory mechanisms to ensure longevity. Continued research into the physiological roles of LL-RNAs in mature neurons and their impact on neuronal longevity will provide valuable insights into the molecular mechanisms underlying neuronal health and aging.