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PUMILIO hyperactivity drives premature aging of Norad-deficient mice

Florian Kopp, Mehmet Yalvac, Beibei Chen, He Zhang, Sungyul Lee, Frank Gillett, Mahmoud Elguindy, Sushama Sivakumar, Hongtao Yu, Yang Xie, Prashant Mishra, Zarife Sahenk, Joshua T Mendell

Preprint posted on October 01, 2018 https://www.biorxiv.org/content/early/2018/10/01/432112

Article now published in eLife at http://dx.doi.org/10.7554/elife.42650

An (un)expected surprise? NORAD lncRNA regulates tissue homeostasis, genome integrity and mitochrondrial function in vivo.

Selected by Carmen Adriaens

Introduction/background

After the realization that a substantial amount of the transcribed genome does not code for proteins, long non-coding RNAs have been an intense subject of study in the past couple of decades. However, still few have been shown to be essential in vivo, and even less are really functionally and mechanistically understood. One of the more recently studied lncRNAs is Noncoding RNA activated by DNA damage, NORAD, named after its induction upon doxorubicin treatment. NORAD has been initially shown to interact with Pumilio proteins involved in post-transcriptional regulation of RNA targets in the cytoplasm. With its 15+ conserved Pumilio Response Elements (PREs), NORAD was shown to be the preferential binding partner of these proteins, thus serving as a decoy (much like lncRNA can serve as a decoy for microRNAs), so that other PUMILIO targets are not downregulated when they are needed in the cell1,2. An important subset of these targets are mRNAs involved in processes such as chromosomal segregation in cell division and DNA replication1,2. When NORAD is lost, Pumilio proteins become hyperactive and excessively inhibit these targets thus causing aberrant mitosis and aneuploidy. Another recent study has identified NORAD as a central scaffold of a novel nuclear complex, NARC1 (NORAD Activated Ribonucleoprotein Complex 1), which is built on the RNA binding protein RBMX3. Upon DNA damage, NARC1 is formed by shuttling NORAD from the cytoplasm back to the nucleus, where it tethers Topoisomerase I and other factors important for genomic stability together with RBMX.

Although a variety of functions and binding partners of NORAD have been investigated by different groups using cultured mammalian cells1–5, its functions have so far not been explored in vivo. In a recent preprint, the group of Prof. J.T. Mendell tackled the question of whether Norad was also, and similarly, functional in living tissues by making a mouse model constitutively deleted for the mouse Norad ortholog, and comparing the phenotypes in these mice with those overexpressing PUM2.

What are the main findings?

First, the authors found that although Norad KO mice were viable and developed initially in a similar fashion as their WT littermates, soon after they reached adulthood they showed signs of premature aging. For instance, they observed increased baldness, greying hairs, spine deformations, weight loss and muscle and neuronal malfunction in Norad KO mice, and noted they had shorter lifespans as compared to wild types. When looking at the molecular cause for these phenotypes, they initially focused their attention to the well-studied interaction of Norad with Pumilio proteins, which had previously been implicated in aging. The authors could indeed identify a conserved interaction between Norad and Pum2, and found that the published canonical Pumilio targets were largely the same in vivo as they were in cultured cells. Furthermore, Pumilio occupancy on its targets was significantly increased in Norad KO mice, which led to a decrease in their expression in this model.

Next, the authors assessed if the aging phenotypes were due to aberrant mitosis previously reported in vitro. For this, they assessed the highly proliferative blood lineage and could indeed establish that both lymphocytes and splenocytes displayed increased aneuploidy.

Because muscle cells were heavily affected by Norad loss and the observed phenotypes pointed to aging-related diseases, the authors assessed if mitochondrial function was affected (for instance, it is known that mitochondrial defects can lead to premature aging6). They noted that Norad KO mice displayed severe mitochondrial abnormalities, including in their morphology and function, which led to a pathological accumulation of reactive oxygen species and oxidative damage, as well as faulty cellular metabolism and decreased mitochondrial respiration rates. Upon further investigation, they found that Norad-depleted tissues and cell lines showed significantly decreased levels of mitochondria-related genes, a subset of which were shown in PUM2 CLIP experiments to be direct targets of this protein.

As Norad was known to tether away Pum2 to prevent unwanted downregulation of its mRNA targets, the authors hypothesized that Norad KO phenotypes could be phenocopied upon Pum2 overexpression if it functioned in the same way in tissues as it does in vitro. Thus, to definitively establish the link between the Norad KO observed phenotypes and Pum2 hyperactivity, they created a mouse model in which doxycycline (dox) administration ubiquitously induced Flag-PUM2 expression. In this model, dox induction did not cause a significant increase in the levels of PUM2 protein, but rather seemed to replace endogenous protein with the tagged version, an observation potentially explained by the known tight regulation of PUM proteins via negative and positive feedback loops. When PUM2 was forcibly expressed, the mice neatly phenocopied, but with an even faster onset, the absence of Norad, both on the gross physiological level and on the cell biological level including the mitochondrial dysfunction.

Overall, the data in this preprint make a compelling case for Norad lncRNA function in adult tissue physiology through interactions with a tightly regulated RNA binding protein.

What do I think about the work?

In short: I really, really like it. It’s a well-controlled study with a beautiful design and compelling results in living animals. I think it is eye-opening that this abundant lncRNA can have such late-onset phenotypes, and I believe it urges the research community to look further than just the binary “viable or not” question. As this study shows, very highly conserved proteins can undergo lineage-specific fine tuning by less conserved RNAs (eg. here, in the mammalian lineage). Furthermore, I am always captivated by the idea that the biology of adult tissues can differ so significantly from these same tissues during development (why do the phenotypes only occur upon reaching adulthood? What changes are made, for instance in the chromatin, that cells become more sensitive to replication stress, and how are developing tissues protected from it when they need to quickly proliferate?).

Some more topical questions, of course, always remain. For instance, in light of the recent study by Munschauer et al., is the NARC1 conserved in mouse tissues, and if so, are any of the observed phenotypes related to NARC1 and the DNA damage regulating factors bound within it? The authors also mention that not all Norad KO mice show the same degree of phenotype. It would be interesting to determine what drives the penetrance of such phenotype, and if in tissues or individuals in which it is less severe, compensatory mechanisms can be identified. Finally, and this maybe just out of curiosity, are Norad KO mice more cancer prone as well? For instance, are blood cancers more prevalent due to the increased aneuploidy, and could this be an important cause of early death?

Ps. I initially named this paper when I saved its PDF “Norad KO pumilio gone mad”. Maybe not appropriate, but since I still like it as a title, I’ll make it my bottomline! :- ).

References

  1. Lee, S. et al. Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins. Cell (2016). doi:10.1016/j.cell.2015.12.017
  2. Tichon, A. et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat. Commun. (2016). doi:10.1038/ncomms12209
  3. Munschauer, M. et al. The NORAD lncRNA assembles a topoisomerase complex critical for genome stability. Nature 561, 132–136 (2018).
  4. Spiniello, M. et al. HyPR-MS for multiplexed discovery of MALAT1, NEAT1, and NORAD lncRNA protein interactomes. J. Proteome Res. 17, 3022–3038 (2018).
  5. Tichon, A., Perry, R. B. T., Stojic, L. & Ulitsky, I. SAM68 is required for regulation of pumilio by the NORAD long noncoding RNA. Genes Dev. (2018). doi:10.1101/gad.309138.117
  6. Trifunovic, A. et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature (2004). doi:10.1038/nature02517

 

 

 

Tags: aging, lncrna, mitochondria, mouse models, norad

Posted on: 9th October 2018

doi: https://doi.org/10.1242/prelights.5136

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