Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X

Colognori, David; Sunwoo, Hongjae; Kriz, Andrea J.; Wang, Chen Yu; Lee, Jeannie T.

doi:10.1016/j.molcel.2019.01.015

Cited by 143 publications

(212 citation statements)

References 81 publications

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“…4b). The eCLIP sites of human HNRNPK protein, whose mouse homologue mediates recruitment of Polycomb through the B and C regions of mouse Xist 29,38 , closely mirror RNP-MaP site density across the human XIST D region and overlap human XIST B and C regions ( Fig. S8), supporting a model wherein the B, C, and D regions of Xist/XIST are functionally analogous 39 .…”

Section: Rnp-map Identifies Conserved Protein Interaction Network Inmentioning

confidence: 65%

“…Strikingly, regions with high RNP-MaP site density in Xist/XIST corresponded to the A, B, C, D, and E regions ( Fig. 4a), which are each known to contain distinct repetitive sequences important for Xist localization, assembly, and chromatin silencing [28][29][30][31][32][33][34][35] . High RNP-MaP density was observed at each of these repetitive regions despite changes in copy number (human XIST contains two copies of the B region) and changes in size, relative position, and sequence (C, D, and E regions differ extensively between mice and humans) 36 .…”

Section: Rnp-map Identifies Conserved Protein Interaction Network Inmentioning

confidence: 99%

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RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Weidmann¹,

Mustoe²,

Jariwala³

et al. 2020

Preprint

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RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but these networks are currently impossible to examine in a comprehensive way.We developed a live-cell chemical probing strategy for mapping protein interaction networks in any RNA with single-nucleotide resolution. This RNP-MaP strategy (RNP network analysis by mutational profiling) simultaneously detects binding by and cooperative interactions involving multiple proteins with single RNA molecules. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein interaction network hubs identify function-critical sites in these RNAs. RNP-MaP identified numerous protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and distinguished communities of proteins that network together on XIST. RNP-MaP data show that the Xist E region is densely networked by protein interactions and that PTBP1, MATR3, and TIA1 proteins each interface with the XIST E region via two distinct interaction modes; and we find that the XIST E region is sufficient to mediate RNA foci formation in cells. RNP-MaP will enable discovery and mechanistic analysis of protein interaction networks across any RNA in cells. RESULTS RNP-MaP ValidationTo comprehensively map protein interaction networks of an RNA of interest, we identified a cellpermeable reagent, NHS-diazirine (SDA), that can rapidly label RNA nucleotides at sites of protein binding. SDA has two reactive moieties: a succinimidyl ester and a diazirine ( Fig. 1a).Succinimidyl esters react to form amide bonds with amines such as those found in lysine side chains. When photoactivated with long-wavelength ultraviolet light (UV-A, 365 nm), diazirines form carbene or diazo intermediates 10 , which are broadly reactive to nucleotide sugar and base moieties. The two-step reaction of SDA thus crosslinks protein lysine residues with RNA with a distance governed by SDA linker length (4 Å) and lysine flexibility (6 Å). Lysine is one of the most prevalent amino acids in RNA-binding domains 11 , and photo-intermediate lifetimes are short; thus, SDA is expected to crosslink short-range RNA-protein interactions relatively independently of local RNA structure or protein properties. To perform crosslinking, live cells are treated with SDA for a short time and then exposed to UV-A light.To detect SDA-mediated RNA-protein crosslinks, we use the MaP reverse transcription technology ( Fig. 1b). SDA-treated cells are lysed and crosslinked proteins are digested to short peptide adducts. Adduct-containing RNA is then used as a template for relaxed-fidelity reverse transcription 12,13 . Under MaP conditions, reverse transcriptase reads through adduct-containing nucleotides and incorporates non-templated nucleotides into the product DNA at the site of RNAprotein crosslinks. Importantly, because transcription reads through adducts, RNP-MaP detects multiple pro...

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Section: Rnp-map Identifies Conserved Protein Interaction Network Inmentioning

confidence: 65%

Section: Rnp-map Identifies Conserved Protein Interaction Network Inmentioning

confidence: 99%

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Weidmann¹,

Mustoe²,

Jariwala³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…22 Another study reported that the repeat B domain within Xist is important for the recruitment and spreading of PRC1 and PRC2 along the Xi and that Xist, PRC1 and PRC2 interdependently regulate X chromosome inactivation. 23 Taken together, these findings prove a clear RNA-mediated function of Xist and establish this lncRNA as an exemplary nuclear RNA that regulates local gene expression.…”

Section: Gene Regulatory Functions Of Lncrna Loci In the Nucleusmentioning

confidence: 72%

“…HDAC3‐mediated histone deacetylation and PRC1‐associated histone H2A ubiquitination have been identified as the earliest chromatin modifications during X chromosome silencing, followed by PRC2‐dependent histone 3 lysine 27 trimethylation (H3K27me3) . Another study reported that the repeat B domain within Xist is important for the recruitment and spreading of PRC1 and PRC2 along the Xi and that Xist , PRC1 and PRC2 interdependently regulate X chromosome inactivation . Taken together, these findings prove a clear RNA‐mediated function of Xist and establish this lncRNA as an exemplary nuclear RNA that regulates local gene expression.…”

Section: Gene Regulatory Functions Of Lncrna Loci In the Nucleusmentioning

confidence: 79%

Molecular functions and biological roles of long non‐coding RNAs in human physiology and disease

Kopp

2019

The Journal of Gene Medicine

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The human genome has been demonstrated to be pervasively transcribed, with a large fraction of the transcriptome representing non‐protein‐coding RNA species. A relatively novel class of non‐coding RNAs, referred to as long non‐coding RNAs (lncRNAs), has attracted increasing attention over recent years. Long non‐coding RNAs comprise a very heterogenous class of transcripts that exert various functions in mammalian cells. Although the number of identified and annotated lncRNAs has been increasing steadily, our understanding of the biological roles for the vast majority of these transcripts remains limited. In this review, a broad concept of the currently known lncRNA modes of action is provided. Examples of mammalian lncRNAs with well‐established biological roles are highlighted and their molecular functions and mechanisms are discussed in detail.

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“…This transcript is expressed from the inactive X chromosome in female cells and coats the Xi chromosome in cis to silence most genes (Galupa & Heard, 2015). XIST RNA is tethered to the Xi chromosome during interphase through a combination of factors including Ciz1 (Ridings-Figueroa et al, 2017;Sunwoo, Colognori, Froberg, Jeon, & Lee, 2017), hnRNP-K (Colognori, Sunwoo, Kriz, Wang, & Lee, 2019;Pintacuda et al, 2017), and hnRNP-U/SAF-A (Hasegawa et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Prophase removal of chromosome-associated RNAs facilitates anaphase chromosome segregation

Wang

Blower

2019

Preprint

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During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A:RNA complexes on mitotic chromosomes and elevated rates of anaphase segregation defects. This work reveals a role for Aurora-B in removing chromatinassociated RNAs during prophase, and demonstrates that Aurora-B dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

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Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X

Cited by 143 publications

References 81 publications

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

Molecular functions and biological roles of long non‐coding RNAs in human physiology and disease

Prophase removal of chromosome-associated RNAs facilitates anaphase chromosome segregation

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