Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

Basu, Reelina; Lai, Lan-Tian; Meng, Zhenyu; Wu, Jun; Shao, Fangwei; Zhang, Li Feng

doi:10.1371/journal.pone.0107425

Cited by 11 publications

(8 citation statements)

References 18 publications

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“…We found a poor overlap with the interactomes of paraspeckles and stress-granules (HotAir: 0 out of 6 are Neat1 interactors, p-value=1 and 0 out of 6 are stress-granules components, p-value=1; Malat1: 5 out of 26 are Neat1 interactors, p-value=0.6, and 7 out of 26 are stress-granules components; p-value=0.2, Fisher's test). HotAir and Neat1 interactomes are significantly smaller than Xist's, supporting the observation that these RNAs form puncti assemblies 33,34 . These observations may indicate that not all lncRNAs are enriched in granule-forming proteins, possibly depending on RNA length, spatial conformation and protein interactions.Structural disorder and nucleic acid-binding are properties of proteins that assemble into ribonucleoprotein (RNP) granules 23 .…”

supporting

confidence: 66%

“…We note that the slight discrepancy in size of ribonucleoprotein assemblies is due to the diverse functions and compositions 4,19,32 . Other lncRNAs such as HotAir, Malat1 or Airn seem to form smaller puncti assemblies, rather than granule-like structures 33,34 . However, a stringent comparative analysis of these lncRNAs and their interacting proteins, by means of super-resolution microscopy is still missing, hampering a more formal comparison.Supporting our observation that Xist assemblies are granule-like, we report that Xist interactions involve a copious number of proteins that phase separate.…”

mentioning

confidence: 98%

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Xist IncRNA forms silencing granules that induce heterochromatin formation and repressive complexes recruitment by phase separation

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Armaos

et al. 2018

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order chromatin structure, Polycomb Repressive Complexes 1/2 (PRC1/2), nuclear matrix, RNAprotein interaction, RNA secondary structure, ribonucleoprotein (RBN). Main textLong non-coding RNAs (lncRNAs) are RNA molecules longer than 200 bases that lack coding potential 1,2 . They represent a significant portion of the cell transcriptome 3 and work as activators or repressors of gene transcription acting on different regulatory mechanisms 4-6 . Indeed, lncRNAs can act as macro-scaffolds for protein recruitment 7-14 and behave as guides and sponges for titrating RNA and proteins, influencing transcription at regulatory regions or triggering transcriptional interference [15][16][17] .The ability of RNA to scaffold protein interactions has been shown to contribute to the formation of membrane-less organelles such as paraspeckles 18 and stress granules 19 . These assemblies, mainly composed of RNAs and proteins, aggregate through a process of liquid-liquid phase separation [18][19][20] . Formation of ribonucleoprotein granules is an evolutionary conserved mechanism for cells to respond to environmental changes 61 and favors the confinement of enzymes and nucleic acids in discrete regions of the nucleus or cytoplasm 21 . Structurally disordered and nucleic acid binding domains promote protein-protein and protein-RNA interactions in the granules 22 .Especially intrinsically disordered proteins, which are enriched in polar amino acids such as arginine, glycine and phenylalanine, have been shown to promote phase transitions in the cell 23 .Recent experiments unveiled an interesting link between the process of phase separation and heterochromatinization [24][25][26] . Indeed, several lncRNAs such as HotAir 27 , ARNIL 28 , Airn 29 and Xist 4 lncRNAs can induce heterochromatin formation to different extent (reviewed in Long et al. 6 ). Xist in particular, the master regulator of X chromosome inactivation, has been shown to induce largescale heterochromatinization of the entire X-chromosome 4 accumulating in large granule-like assemblies that can be investigated by super-resolution microscopy 30,31 ( Fig. 1A). Female cells show ~100-150 Xist-containing complexes 30,31 that largely resembles paraspeckles and stress granule in size and protein composition (see below). Intriguingly, Xist assemblies measure around 100-200 nm in diameter 31 , comparable with stress granules and paraspeckles dimensions (~100-300 nm and ~200-500 nm in diameter, respectively 32,19 ) ( Fig. 1A). We note that the slight discrepancy in size of ribonucleoprotein assemblies is due to the diverse functions and compositions 4,19,32 . Other lncRNAs such as HotAir, Malat1 or Airn seem to form smaller puncti assemblies, rather than granule-like structures 33,34 . However, a stringent comparative analysis of these lncRNAs and their interacting proteins, by means of super-resolution microscopy is still missing, hampering a more formal comparison.Supporting our observation that Xist assemblies are granule-like, we report that Xist interactions involve a copious nu...

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