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

Abstract

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...

Structural modularity of the XIST ribonucleoprotein complex

“…Multivalent interactions underlie phase separation, a general mechanism of organization in cells. Recently it was reported that the XIST RNP complex forms a distinct liquid phase in cells, probably due to the repetitive sequences in XIST and the intrinsically disordered regions in XIST-associated proteins (Cerase et al, 2018). Our analysis showed that XIST associated proteins bind XIST in clustered patterns, supporting the multivalent interactions.…”

The Role of Long Non-Coding RNAs in Cardiovascular Diseases

“…Xist will cause the X chromosome in its place to relocate to the outer edge of the nucleus. Afterwards, it interacts with PRC2 to elevate H3K27me3, resulting in X chromosome silencing and heterochromatinization [18,19] (3) HOTTIP recruits WDR5 to the promoter region of HOXA, the critical developmental gene, to activate its expression [20,21]. In addition, lncRNAs can block transcription factors from binding to the target region (Figure 2c).…”

lncRedibly versatile: biochemical and biological functions of long noncoding RNAs

“…Finally, it has been recently hypothesized that XIST might silence the X chromosomes by forming phase-separated "silence granules", due to its interactions with disordered proteins, including FUS and hnRNPA2 [22,153]. Although experimental evidence is required to support this suggestion, it provides an interesting new perspective on the biochemistry of XIST-mediated silencing.…”