Project 1: NEAT1

An architectural long non-coding RNA, NEAT1, presents a paradox: three experimentally confirmed mammalian orthologs do not exhibit sequence similarity but retain full functionality—the paraspeckles that they form were detected in all three species, and the main resident proteins were identified. So, how is it possible to preserve functionality and structure without maintaining sequence homology? In this paper, we addres this question by applying a comparative phylogenetic approach.

The uniqueness of our method lies in the joint study of two syntenically connected lncRNAs—NEAT1 and MALAT1—which also share a very unique maturation process involving the tRNA-processing machinery. As a result, we were able to identify NEAT1 orthologs across a wide range of mammals, even when primary sequence similarity was absent.

We found that certain structural features, like tRNA-like structures, triple helices, and specific protein-binding sites, are well-preserved over time. In NEAT1, we also identified stable regions that help it interact with important proteins like TDP-43 and NONO. Our results suggest that the shorter form of NEAT1 has a distinct role, as it is more conserved in both sequence and length. We also found evidence that the process controlling which NEAT1 form is produced may be similar across species, likely mediated by TDP-43. Additionally, we showed that repetitive DNA elements have played a key role in NEAT1’s evolution by affecting its length, creating stable structures, and introducing new protein-binding sites.

Project 2: Paraspeckles

Mammalian species not only exhibit distinct embryonic development timelines but also display remarkable variations in lifespan. We seek to determine whether these differences in lifespan manifest at the cellular level. Cellular stress is considered a key trigger in cellular aging, and we hypothesize that species with longer lifespans may exhibit greater cellular stress resistance.

Paraspeckles are membraneless organelles found exclusively in mammals, which are primarily composed of the long non-coding RNA NEAT1 and various RNA-binding proteins (RNPs). Studies have shown that paraspeckles are closely associated with cellular stress responses. We propose that the stress resistance capacity of paraspeckles may vary across species and that this variation could influence species lifespan.

To compare the stress resistance capacity of paraspeckles across different species, we plan to use CRISPR to knock out the Neat1 gene in mouse embryonic stem cells. Additionally, we will employ recombineering technology to construct a donor plasmid carrying the human NEAT1 gene and use the Flp-in system to replace the endogenous Neat1 gene with its human counterpart. Subsequently, we will induce differentiation of these cells into neural cells and apply various stress stimuli to mimic the cellular aging process. Finally, by analyzing paraspeckle number, cell survival rate, and metabolic activity, I aim to compare the stress resistance capacity of paraspeckles across different species.

Project 3: Metabolism and species specific development rates

Embryos from different mammalian species develop at characteristic timescales. These timescales are recapitulated during the differentiation of pluripotent stem cells in vitro. Specific genes and molecular pathways that modulate cell differentiation speed between mammalian species remain to be determined. Here we use single-cell multi-omic analysis of neural differentiation of mouse, cynomolgus and human pluripotent cells to identify regulators for differentiation speed.

We demonstrate that species-specific transcriptome dynamics are mirrored at the chromatin level, but that the speed of neural differentiation is insensitive to manipulations of cell growth and cycling. Exploiting the single-cell resolution of our data, we identify glycogen storage levels regulated by UDP-glucose pyrophosphorylase 2 (UGP2) as a species-dependent trait of pluripotent cells, and show that lowered glycogen storage in UGP2 mutant cells is associated with accelerated neural differentiation. The control of energy storage could be a general strategy for the regulation of cell differentiation speed.

Project 4: Mammalian Stem Cell Zoo

In addition to the species-specific timescales in embryonic development different mammalian species show vastly different lifespans. We wonder if these different lifespans are reflected at a cellular level. We hypothesize that the efficiency of stress response mechanisms of non-dividing cells is higher in mammals with a long lifespan. It is apparent that testing this hypothesis within the context of living organisms is impossible. However, the production of species-specific induced pluripotent stem cells (iPSCs) from non-invasive or post-mortem biopsies could enable the in vitro study of our hypothesis. We are consistently expanding the panel of available pluripotent stem cells from different species in our lab.

To compare the resistance to cellular stress between these species, we differentiate these cells into terminal neuronal cell types. In a high-throughput format we are establishing the use of low dosages of small-molecules to stress these neuronal cells repeatedly, mimicking aging at a cellular level. We aim to measure the survival and stress responses of these neuronal cells with time-lapse microscopy, utilizing live/dead staining and potentially fluorescent reporters for specific cellular stress pathways.

Project 5: Census of iPSC reprogramming methods for understudied mammalian species

The ability to generate induced pluripotent stem cells (iPSCs) from diverse species represents an exciting opportunity to study the molecular and cellular basis of interspecies differences across a wide range of phenotypes, from development to lifespan and ageing. iPSCs lines from numerous species have been generated over the past 10 years, but efficient use of these resources is still hampered by the lack of a coherent database that collates critical information about existing lines, such as culture conditions, pluripotency state, and availability.

We have started a community effort to build such a database, where we have collected information about more than 150 stem cell lines so far. We plan to publish this database as a public resource in the near future, accompanied by a review about emerging applications of pluripotent stem cells from understudied species.