Functional conservation of the lncRNA NEAT1 in the ancestrally diverged marsupial lineage: Evidence for NEAT1 expression and associated paraspeckle assembly during late gestation in the opossum Monodelphis domestica.

Long non-coding RNAs (lncRNAs) are widely expressed and play various roles in cell homeostasis. However, because of their low conservation at the sequence level, recapitulating lncRNA evolutionary history is often challenging. While performing an ultrastructural analysis of viral particles present in uterine glands of gestating opossum females, we serendipitously noticed the presence of numerous structures similar to paraspeckles, nuclear bodies which in human and mouse cells are assembled around an architectural NEAT1/MENϵ/β lncRNA. Here, using an opossum kidney (OK) cell line, we confirmed by immuno-electron microscopy the presence of paraspeckles in marsupials. We then identified the orthologous opossum NEAT1 gene which, although poorly conserved at the sequence level, displays NEAT1 characteristic features such as short and long isoforms expressed from a unique promoter and for the latter an RNase P cleavage site at its 3'-end. Combining tissue-specific qRT-PCR, in situ hybridization at the optical and electron microscopic levels, we show that (i) NEAT1 is paraspeckle-associated in opossum (ii) NEAT1 expression is strongly induced in late gestation in uterine/placental extracts (iii) NEAT1 induction occurs in the uterine gland nuclei in which paraspeckles were detected. Finally, treatment of OK cells with proteasome inhibitors induces paraspeckle assembly, as previously observed in human cells. Altogether, these results demonstrate that paraspeckles are tissue-specific, stress-responding nuclear bodies in marsupials, illustrating their structural and functional continuity over 200 My of evolution throughout the mammalian lineage. In contrast, the rapid evolution of the NEAT1 transcripts highlights the relaxed constraint that, despite functional conservation, is exerted on this lncRNA.