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 - PubMed (original) (raw)

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

Guillaume Cornelis et al. RNA Biol. 2016 Sep.

Abstract

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.

Keywords: Immuno-electron microscopy; NEAT1; lncRNAs; nuclear bodies; opossum; paraspeckles; pregnancy; ultrastructure; uterine glands.

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Figures

Figure 1.

Paraspeckle-like structures as observed in opossum uterine glands by conventional EM. (A) Left frame. Hematoxylin eosin saffron (HES)-stained section showing cellular organization of placental and uterine tissues during late gestation (12dpc). The placental syncytiotrophoblast layer (St) is in direct contact with the maternal uterine epithelium (Ue). Underneath the uterine epithelium, uterine glands (Ug) form rings, with heavily-stained peripheral cell nuclei. L: lumen. Right frame. EM micrograph showing dense paraspeckle-like nuclear structures (arrows) as observed in ultra-thin sections of opossum uterine glands at the same stage. Notice the compact fibrillar structure (inset). Ch: peripheral heterochromatin. Scale bars: 20, 2 and 0.2 µm respectively. (B) Micrographs showing paraspeckle-like fibrillar structures that are (i) tightly-associated with Interchromatin Granules (IG) (ii) cylindrical (iii) in clusters. Scale bars: 0.2 µm. (C) Values of the short (Sx) and long axis (Lx) of 31 paraspeckle-like structures (as measured in B, right frame), when plotted by increasing Lx, illustrate variable length and constant diameter (mean Sx 295 +/− 35 nm). Among nuclear bodies in human cells, this elongation profile is distinctive of paraspeckles.

Figure 2.

OK cells contain NEAT1-associated paraspeckle nuclear bodies that are induced by proteasome inhibition. (A) IF of control and MG132-treated (5 µM, 6h) OK cells (left and right frames respectively) stained with an anti-NONO antibody (green) and counterstained with DAPI (DNA, blue) reveals scattered fluorescent foci in nuclei of untreated OK cells and their increased frequency upon proteasome inhibition (arrow-heads). Scale bars: 10 µm. (B) Ultrastructural characterization of opossum paraspeckles by I-EM with antibodies against human DBHS proteins. Ultrathin sections of Lowicryl-embedded MG132-treated OK cells were incubated with the indicated primary antibodies and secondary antibodies conjugated to 10 nm gold particles. Labeled paraspeckles are shown. IG: Interchromatin granules. Scale bars: 100 nm. (C) A biotinylated opossum NEAT1 5′-end DNA probe (see below) hybridized onto ultrathin sections of Lowicryl-embedded MG132-treated OK cells detects NEAT1 RNA 5′-end at the paraspeckle periphery. DNA/RNA hybrids were detected with an anti-biotin antibody conjugated to 10 nm gold particles. Scale bars: 100 nm.

Figure 3.

Homology search of NEAT1 in the opossum genome. (A) Alignment of syntenic FRMD8-SCYL1 region in human, mouse and opossum genomes shows significant DNA sequence conservation at the protein coding FRMD8 and SCYL1 and non coding MALAT1 genes but not within the presumptive NEAT1 region. RNase P cleavage sites (P) at the 3′-end of the human NEAT1 and _MALAT_1 genes and at the 3′-end of the opossum MALAT1 gene are underlined (arrow-head). (B) Comparison of opossum and Tasmanian Devil (TD) marsupial genomes highlights an homologous repeat-free DNA sequence downstream of FRMD8 (star) and, within TD but not opossum DNA, an RNase P site corresponding to the NEAT1_2 3′-end. (C) Mapping of the RNase P processed 3′-end of the opossum NEAT1 gene by sequencing of un-annotated regions. The location on chromosome 8 of the opossum genome (UCSC database) of 3 gaps that were PCR-amplified and sequenced is shown as red boxes. The triple-helix forming motifs (green) and modeled structure of the downstream tRNA-like element found in gap 3 are depicted. The putative RNase P and RNase Z cleavage sites at the 3′-end of the opossum NEAT1_2 isoform are inferred by comparison with the tRNA-like structure in the human locus.

Figure 4.

Delineating the opossum NEAT1_1 transcription unit by RACE. (A) A single NEAT1 transcription initiation site was mapped by 5′-RACE on cDNA from total RNA extracted from a mixed uterine/placental sample at 13 dpc. Conservation of proximal DNA sequence in marsupials is illustrated. TATA box is underlined by a red square. Positions of qPCR, riboprobes and DNA probe primers are depicted. (B) Two polyadenylated NEAT1_1 3′-ends downstream of canonical (AATAAA) PAS were uncovered by 3′-RACE from the same RNA sample. The last nucleotide preceding the poly-A tail is indicated for both 3′-ends. Different DNA sequence conservation of the NEAT1_1a and NEAT1_1b PAS within the marsupial and eutherian genomes is shown in red boxes. (C). Relative abundance of the 2 NEAT1_1 opossum isoforms within oligo-dT-primed 3′-RACE cDNA from a uterine/placental sample at 13 dpc. Positions of qPCR primers specific of the NEAT1 1_1b or 1_1a + 1_1b isoforms are depicted in (B). Relative abundance (relative to the amount of the housekeeping RPLP19 gene) +/- SEM was determined from duplicate samples in 2 independent qPCR experiments.

Figure 5.

Real-time RT-qPCR analysis of the opossum NEAT1 transcripts in various tissues.Transcript levels are expressed as the ratio of their expression level to that of the RPL0 control gene (see Methods). Due to the high interpenetration of maternal and fetal tissues, placental and uterine tissues are analyzed as a whole at 3 gestational times (12, 13 and 14 dpc). Values are the means of duplicates from 3 samples ± SEM, except for the non pregnant uterus, the mammary gland and embryo samples, where only one sample was analyzed. Dotted line indicates the ratio value equal to 1. Location of primers with respect to NEAT1 isoforms is indicated in Fig. 4A.

Figure 6.

NEAT1_2 expression in opossum pregnant uterine glands. Serial sections of opossum pregnant uterus stained with (A) HES, (B-D) using digoxigenin-labeled NEAT1_2 sense (B) or antisense (C) riboprobes revealed with an alkaline phosphatase-conjugated antidigoxigenin antibody. (D) Enlargement of the antisense riboprobe staining. Specific staining is observed at the level of the nuclei of the uterine gland cells, while the uterine epithelium is unlabelled. Location of riboprobes is indicated in Fig. 4A. (A-C): scale bar: 50 μm. (D): scale bar: 10µm.

Figure 7.

Paraspeckle formation predates divergence of marsupial and eutherian mammals. (A) Paraspeckle emergence at least 200 Mya in a common ancestor to eutherian and marsupial mammals is suggested by their structural conservation in human, mouse and opossum cell nuclei. Micrograph scale bars: 100 nm. (B) Level of NEAT1 identity between species was defined by measuring the % of sequence aligned (cov) and the % of identity within aligned segments (id), using BLASTN. These values are reported on the graph with the identity color code shown on the left scale to underline the higher conservation of NEAT1_1 as compared to NEAT1_2 and the overall low conservation of both NEAT1 isoforms between marsupial and eutherian mammals.

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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 - PubMed (original) (raw)