Systematic discovery of Xist RNA binding proteins

Abstract

Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.

Figure 1. ChIRP-MS method and validation

(A) outline of the ChIRP-MS workflow. Briefly, RNP complexes are crosslinked in vivo by 3% formaldehyde for 30min, and solubilized by sonication. Target ncRNA are pulled out by biotinylated anti-sense oligos, and associated proteins are eluted with free biotin, separated by electrophoresis, and each size fraction is subjected to LC/MS-MS identification. (B) Distribution of input, and U1 and U2-enriched RNA sizes, as determined by Bioanalyzer (Agilent). (C) Proteins retrieved by U1, U2, U3 and control probes, analyzed by immunoblotting. Arrow indicates the U1A close homolog, U2B, cross-identified by U1A antibody. (D) Proteins retrieved by U1, U2, Rnase-treated controls and non-targeting probe control, visualized by silver staining. Major proteins enriched are indicated on the left.

Figure 2. U1/U2 ChIRP-MS

(A) Venn diagram of known spliceosome proteins, and proteins pulled-down by U1 or U2. The number of interactions in each set is given after the set label. (B) Numbers of U1/U2 pulled-down proteins by their degrees of separations from known spliceosome proteins. The dashed line represents the distribution of a randomly simulated set of the same number of proteins pulled-down by U1 and U2 (right axis). (C) Protein-protein and protein-RNA interaction network of U1/U2 pulled-down proteins. Proteins belonging to known complexes are organized and annotated in groups in top half of the plot, and proteins of unknown affiliation are presented at the bottom. Complexes and proteins more strongly enriched by U1 (left in graph) (e.g. Polyadenylation and cleavage, Nop56p) are positioned accordingly.

Figure 3. Xist ChIRP-MS

(A) >60% of Xist RNA was retrieved from the cell by ChIRP, while no Gapdh was detected. RNase treatment eliminates Xist transcripts prior to pulldown. (B) Proteins retrieved by Xist and isogenic control (no Xist), visualized by Coomassie blue staining. (C) Validation of ChIRP-enriched proteins by immunoblotting.

Figure 4. Xist partner proteins are developmentally regulated

(A) Heatmap of Xist-RBPs pulled down in the four experiments. Color bars indicate abundance of peptides detected. Protein annotations were color-designated based on their class. (B) Similar proteins are enriched between differentiating ES cells vs. EpiSCs and (C) between EpiSCs and TSCs.

Figure 5. Functional characterization of Xist RBPs

(A) Relative positions of Grb10 and Xist transgene (TG) on chr11. (B) Induction of Xist and repression of Grb10 by different doses of dox in e36 cells that have undergone RA-induced differentiation for 4 days. (C) Western validation of HnrnpU, K and M knockdown by siRNAs. (D) De-repression of Grb10 upon depletion of HnrnpU, K and M. (E) Dual-color FISH of Grb10 and Xist in e36 cells that are depleted of HnrnpK. Arrowheads indicate Grb10 allele escaping Xist silencing. (F) Quantification of cells with Grb10 expression on the Xist-coated chromosome by counting >150 cells from 3 replicates.

Figure 6. HnrnpK is required for repressive chromatin modifications of inactive X

(A) Northern blot against Xist in e36 cells depleted of HnrnpM, U or K. (B) Xist sm-FISH in HnrnpU and K knockdown cells. (C-D) IF co-FISH of Xist and H3K27Me3 (C)/H2AK119ub (D) in HnrnpK knockdown cells. Number of cells with strong, weak and undetectable repressive marks overlapping with Xist foci were tallied and represented below.

Figure 7. Xist A-repeat binds Spen, a silencing factor that contributes to XCI

(A) Similar proteins are enriched by ChIRP-MS of full-length Xist and A-repeat mutant, except three highlighted proteins: Wtap, Rnf20, and Spen. (B) siRNA depletion of the indicated factors show only Spen is required for X-linked silencing of Pgk1. (C) siRNA depletion of Spen interferes with XCI in cells, as indicated by co-localization of Xist “cloud” and active transcription of X-linked genes Rnf12 and MeCP2 (arrowheads) on the same chromosome. (D) Quantification of cells with expression of Mecp2 and Rnf12 on the Xist-coated chromosome by counting > 100 cells from 3 replicates. A proportion of cells do not upregulate Xist and do not coat (around 40%); we counted only the cells with Xist domains. (E) Model of the cell-state- and scaffold-specific loading of Xist-RBPs, and their chromatin modifying functions.